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					NEWSLETTER OF NOVAGEN, INC. •                   ADVANCED PRODUCTS AND PROTOCOLS FOR MOLECULAR BIOLOGY RESEARCH
                                                                                                                NUMBER 6
                                                                                                              DECEMBER 1996

 FEATURE ARTICLE                                                                                          IN THIS ISSUE


T7Select® Phage Display                                                                                      ARTICLES

System: A powerful new                                                                              T7Select Phage Display System:
                                                                                                    A powerful new protein display

protein display system                                                                             system based on bacteriophage T7
                                                                                                                 1
based on bacteriophage T7                                                                             Perfectly Blunt Cloning:
Alan Rosenberg1, Kathleen Griffin1, F. William Studier1, Mark McCormick2,                           A superior method for cloning
James Berg2, Robert Novy2, and Robert Mierendorf2                                                    PCR products or any DNA
— 1Biology Dept., Brookhaven National Laboratories and 2Novagen, Inc.
                                                                                                                 7

P
         hage display is a powerful tech-        clones, and new applications are continually
         nique for identifying peptides          arising (2).                                           Determine nucleic acid
         or proteins that have desirable             We have developed a new phage display             concentration to 0.25 ng
         binding properties. In this method,     system based on bacteriophage T7. This                   with Spot-On kit
a peptide or protein is displayed on the
surface of a phage as a fusion to a protein
                                                 system is easy to use and has the capacity
                                                 to display peptides up to about 50 amino
                                                                                                                12
that is normally found in the phage particle.    acids in size in high copy number (415 per
The first phage vectors suitable for surface     phage), and peptides or proteins up to about
display were made by Smith and coworkers,        1200 amino acids in low copy number (0.1–1               NEW PRODUCTS
using filamentous phage (1). They also           per phage). T7 is a double-stranded DNA
developed simple procedures for selecting        phage that has been extensively studied (3, 4).                13
phage displaying peptides or proteins that                                 continued on page 2
bind to particular targets. Such phage can
be selected from large libraries of variants.
                                                                                                         ANNOUNCEMENTS

This system is easy to use and has the
                                                                                                                16
capacity to display peptides up to about
50 aa in size in high copy number, and                         Bind
peptides or proteins up to about 1200 aa
in low copy number.

   Both the peptide or protein and its
coding sequence are selected at the same                       Elute
time, since the displayed peptide or protein
responsible for the binding is encoded in
the genome of the bound phage. Phage                         Amplify
display has been used to identify peptides
that bind to receptors, substrates or in-
hibitors of enzymes, epitopes, improved
antibodies, altered enzymes, and cDNA
 FEATURE ARTICLE
continued from page 1
Vector                      Use            Display Number                 Display Limit                     Host              T7Select vectors
                                                                          (amino acids)                                           There are two basic types of T7Select
T7Select®415-1            peptides              415                          40–50 aa                      BL21               phage display vectors: the T7Select415
T7Select1-1         peptides or proteins        ≤1                             900 aa                     BLT5403
                                                                                                                              vector for high-copy number display of pep-
T7Select1-2         peptides or proteins        ≤1                            1200 aa                     BLT5403
                                                                                                                              tides, and the T7Select1 vectors for low-
Table 1. Phage display vector features.                                                                                       copy number display of peptides or larger
                                                                                                                              proteins (Table 1). In all of the vectors,
Phage assembly takes place inside the                 is released and a conformational change                                 coding sequences for the peptides or pro-
E. coli cell and mature phage are released by         occurs in the shell to form the mature parti-                           teins to be displayed are cloned within a
cell lysis. Unlike the filamentous systems,           cle. Tail and tail fibers attach at the head-                           series of multiple cloning sites following aa
peptides or proteins displayed on the surface         tail connector vertex.                                                  348 of the 10B protein (Figs. 2 and 3). The
of T7 do not need to be capable of secretion              The T7Select ® Phage Display System                                 natural translational frameshift site within
through the cell membrane, a necessary step           uses the T7 capsid protein to display peptides                          the capsid gene has been removed, so only a
in filamentous phage assembly (5).                    or proteins on the surface of the phage.                                single form of capsid protein is made from
    T7 has additional properties that make            The capsid protein is normally made in                                  these vectors.
it an attractive display vector. It is very           two forms, 10A (344 aa) and 10B (397 aa).                                   Functional peptides up to 39 amino acids
easy to grow and replicates more rapidly              10B is produced by a translational frameshift                           have been displayed from T7Select415.
than either bacteriophage λ or filamentous            at amino acid (aa) 341 of 10A, and makes                                Expression of the T7Select415 capsid gene
phage. Plaques form within 3 hours at 37 °C
and cultures lyse 1–2 hours after infection,
decreasing the time needed to perform the
multiple rounds of growth usually required
for selection. The T7 phage particle is
extremely robust, and is stable to harsh con-
ditions that inactivate other phage. This
expands the variety of agents that can be
used in biopanning selection procedures,
which require that the phage remain infec-
tive. T7 is actually an excellent general
cloning vector. Purified DNA is easy to
obtain in large amounts, a high-efficiency
in vitro packaging system is available (6),
and the DNA is completely sequenced
(39,937 bp), so restriction or DNA sequence
analysis of clones is quite straightforward.

T7 structure and assembly
   T7 is an icosahedral phage with a capsid
shell composed of 415 copies of the T7
capsid protein (gene 10) arranged as 60
hexamers on the faces of the shell and 11
pentamers at the vertices (4). Attached at
the remaining vertex is the head-tail con-
                                                      Fig. 1. Structure of the T7 phage particle.
nector (gene 8), a short conical tail (genes          The capsid shell, head-tail connector, tail, and tail fibers are shown schematically. The diffraction pattern from polyheads (4) showing a
11 and 12) and 6 tail fibers (gene 17)                hexamer capsid unit has been fit onto the surface of the icosahedral particle (diameter approx. 55 nm). The monomer units are in gray.
(shown schematically in Fig. 1).
   The phage assembly process is similar to           up about 10% of the capsid protein (8).                                 is controlled by the same strong phage pro-
that of other double-stranded DNA phages              However, functional capsids can be com-                                 moter (φ10) and translation initiation site
(7). DNA is packaged into a procapsid shell           posed entirely of either 10A or 10B, or of                              (s10) as in wild-type phage (Fig. 3), and
made up of scaffolding protein (gene 9),              various ratios of the proteins (9). This finding                        the capsid/peptide fusion protein is pro-
capsid protein, the head-tail connector, and          provided the initial suggestion that the T7                             duced in large amounts during infection.
an internal protein structure (genes 13, 14,          capsid shell could accommodate variation,                               T7Select415 clones usually grow well on
15, and16). The DNA is packaged from                  and that the region of the capsid protein                               normal T7 laboratory hosts, such as E. coli
linear concatemers, and as the DNA enters             unique to 10B might be on the surface of the                            BL21. The capsid shell of the phage is com-
the procapsid shell the scaffolding protein           phage and could be used for phage display.                              posed entirely of the capsid/peptide fusion

                                                                                                                                                                         inNovations 6
  2
protein, thereby displaying 415 copies of
peptide on the surface of the phage. High
                                                    T7Select415-1b, T7Select1-1b
copy number display is desirable wherever a                aa348                                          aa363
strong signal is useful, such as in epitope         ...MetLeuGlyAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGlu
                                                    ...ATGCTCGGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGTAACTAGTTAA
mapping. It may also be important for                            BamH I EcoR I Sac I Sal I Hind III Not I Xho I
obtaining peptides that at best bind only
                                                    T7Select1-2a
very weakly to their targets.                              aa348                                                       aa368
    Functional proteins up to slightly more         ...MetLeuGlyGlySerAspIleGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgValThrSer
                                                    ...ATGCTCGGTGGATCCGATATCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGTAACTAGTTAA
than 1000 amino acids have been displayed                        BamH I EcoR V EcoR I Sac I Sal I Hind III Not I Xho I
from T7Select®1-1 vectors. The T7Select1-
2a,b,c series provides multiple cloning             T7Select1-2b
                                                           aa348                                                 aa365
sites in all three reading frames. Peptides or      ...MetLeuGlyAspProIleSerAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGlu
proteins are displayed in low copy number           ...ATGCTCGGGGATCCGATATCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGTAACTAGTTAA
                                                                 BamH I EcoR V EcoR I Sac I Sal I Hind III Not I Xho I
(about 0.1–1 per phage) from these vectors,
which makes them suitable for the selection         T7Select1-2c
                                                           aa348                                                      aa366
of proteins that bind strongly to their tar-        ...MetLeuGlyIleArgTyrArgIleArgAlaProSerThrSerLeuArgProHisSerSerAsn
gets. In order to obtain low-copy display,          ...ATGCTCGGGATCCGATATCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGTAACTAGTTAA
the promoter of the capsid gene was re-                         BamH I EcoR V EcoR I Sac I Sal I Hind III Not I Xho I

moved and the translation initiation site
                                                  Fig. 2. T7Select vector cloning regions.
was altered (Fig. 3). The capsid mRNA is          All vectors contain a multiple cloning site following 10B aa 348. The T7Select1-2a,b,c series contains the cloning sites in all three
still produced from phage promoters located       reading frames and includes a blunt-end site (EcoR V).

further upstream of the gene, but produc-
tion of capsid protein is greatly reduced.       have a good chance of including all variants                          high copy number (415 per phage) from
T7Select1 phage are grown on a comple-           increases with the number of varied amino                             T7Select415 include:
menting host (BLT5403) that provides large       acids. For example, a complete heptapep-                              • S•Tag (15 aa) from pancreatic
amounts of the 10A capsid protein from a         tide library has 207 = 1.28 × 109 unique hep-                           ribonuclease A
plasmid clone. The 10A gene in the com-          tapeptides. The capacity to construct large
                                                                                                                       • HSV•Tag® epitope (11 aa) from
                                                 libraries in any cloning system depends
                                                                                                                         Herpes Simplex Virus glycoprotein D
The phage maintains infectivity follow-          on the overall efficiency of cloning and
                                                 packaging (phage) or transformation (plas-                            • Streptavidin-binding peptide (10 aa) (10)
ing treatment with 1% SDS, 5 M
                                                 mids). The vector arms and T7 packaging                               • RGD peptide (8 aa) from adenovirus
NaCl, up to 4 M urea, 2 M guani-
                                                 extracts provided in the T7Select System                                penton protein (11)
dine-HCl, 10mM EDTA, reducing
                                                 routinely produce > 10 8 recombinant                                  • Thrombin cleavage site (7 aa) from
conditions (up to 100 mM DTT),                   plaques per µg of arms. This efficiency is 10-                          pET vectors
and alkaline conditions (up to pH 10).           to 50-fold higher than usually observed with
                                                                                                                       • HSV•Tag + His•Tag® sequences (39 aa)
                                                 λ cloning systems, and comparable to the
plementing plasmid and the capsid gene in        optimal efficiency of plasmid systems. The                               These peptides were cloned on DNAs
the vectors have been engineered to mini-        high-efficiency T7 packaging extracts (2 ×                            that added from 10–39 aa to the 10B capsid
mize any recombination between the genes.        109 plaques per µg intact DNA) are made                               protein (measured from aa 348, the last nat-
                                                 with a specially designed phage that reduces                          urally occurring aa). In each case, the dis-
Cloning in T7Select vectors                                                                                            play of functional peptide was verified by
                                                 the non-recombinant cloning background
    Procedures for cloning in T7Select           to below 0.1%.                                                        an appropriate binding assay. The use of
vectors are similar to those for cloning in          For verification of performance, the                              the thrombin cleavage site enabled us to
bacteriophage λ vectors. Vector arms are         T7Select Cloning Kits include a positive                              demonstrate directly that all 415 copies of
prepared and ligated with target inserts, the    control target DNA, which encodes the 15 aa                           peptide appear to be on the surface of the
resulting DNA is incubated with an in vitro      S•Tag peptide. S•Tag recombinants are                                phage and can be clipped off by thrombin
packaging extract, and the phage products        easily detected with a rapid, chemilumines-                           without reducing the infectivity of the
are used for infection of a suitable host.       cent plaque lift assay using the T7Select                             phage (Fig. 4).
The multiple cloning sites in the T7 vectors     Biopanning Kit.                                                          Peptides or proteins displayed in low
(Fig. 2) are compatible with many existing                                                                             copy number (0.1–1 per phage) from
vectors, including the pET vectors used in       Examples of peptide                                                   T7Select1 vectors include:
                                                 and protein display
the T7 expression system.                                                                                              • E. coli β-galactosidase (1015 aa)
    The target DNA inserts usually contain          We have displayed a variety of bio-                                • T7 RNA polymerase (873 aa)
a limited region coding for variant amino        logically active peptides and proteins from
acids. The size of the library required to       the T7Select vectors. Peptides displayed in                                                                 continued on page 4

inNovations 6
                                                                                                                                                                                    3
 FEATURE ARTICLE
continued from page 3
                                                                                                                                           displayed in low copy number (0.5 per
                                                                                                                                           phage, Fig. 7) and the phage mixed with
           0                           20                       40                            60               80           100            control phage in a ratio of 1 to 10 6, the
               Early genes            DNA metabolizing genes                    Structure and assembly genes
                                                                                                                                           HSV•Tag phage constituted about 25% of
 T7                                                                                                                                        the population after three rounds of se-
                             1                                                       10
                        T7 RNA pol                                               Capsid                                                    lection. This represents a 50- to 100-fold
                                                                                                                                           enrichment for each round of selection.
                                 φ9                       φ10                        Tφ                                        φ13             The stability of the T7 phage particle
                                            Scaffolding              Capsid                   Tail                  Tail                   enables the use of a variety of elution condi-
                                                9                       10                    11                    12                     tions during biopanning. The phage main-
                                                                                                                                           tains infectivity following treatment with
                                                                                                                                           1% SDS, 5 M NaCl, up to 4 M urea, 2 M
                                                                                                                                           guanidine-HCl, 10 mM EDTA, reducing
                                        φ10                                                          Tφ
                                                                                                                                           conditions (up to 100 mM DTT), and alka-
 WT                                           s10                                                                                          line conditions (up to pH 10). The phage is
                                                                10A                     B
                                                                                                                                           not stable to low pH (below 4 or so), a con-
                                                                                  348       397
                                                                                                                                           dition often used in biopanning for filamen-
                                                                                                                                           tous phage. Biopanning requires both bind-
                                                                                                                                           ing and elution conditions that maintain
                                        φ10                                                    Tφ
                                                                              Cloning                                                      the infectivity of the phage. The wide range
                                                                                 sites
 T7Select415-1                                s10
                                                                                                                                           of conditions available for T7Select ®
                                                                10B
                                                                                                                                           biopanning should expand the range of tar-
                                                                               348      363
                                                                                                                                           gets that can be used successfully. The
                                                                                                                                           T7Select Biopanning Kit provides the mate-
                                                                                               Tφ
                                                                                                                                           rials necessary for testing your own biopan-
                                                                              Cloning
                                                                                 sites                                                     ning procedures, using phage that display
 T7Select1-1, 2-1                                               10B
                                                                                                                                           the S•Tag peptide.
                                                                               348      363
                                                                                                                                           Characteristics of the system
 Fig. 3. Phage display vectors and the genetic map of T7.                                                                                      Large proteins cannot be cloned in
 The T7 capsid gene (gene 10) is located at about position 60 in the T7 genome, within the region of genes coding for proteins             T7Select415, the high copy number display
 involved in the structure and assembly of T7. Capsid protein expression during infection is controlled by a promoter (φ10) and termi-
 nator (Tφ) for T7 RNA polymerase, and by string translation initiation signals (s10). The capsid protein is normally made in two forms,   vector. The largest peptide yet displayed
 10A (344 aa) and 10B (397 aa), related by a translational frameshift at 10A aa 341. The T7Select415 and T7Select1 vectors contain a
 multiple cloning site following aa 348 of a 10B gene that is in a single reading frame, i.e., only the truncated 10B form is made from
                                                                                                                                           from T7Select415 is 39 aa long. It seems
 these vectors. Capsid protein expression from T7Select415 vectors is controlled as in the wild-type phage.                                likely that peptides up to at least 50 amino
                                                                                                                                           acids will work, since this will create a
• scFv single-chain antibody (257 aa)                                           and, where the phage has been purified,                    capsid protein about the same size as the
                                                                                whether the enzymatic activity survives the                wild-type 10B protein. The capacity of
• T7 endonuclease (149 aa)
                                                                                purification. For example, phage displaying                T7Select415 is clearly sufficient for display-
• S•Tag (15 aa)
                                                                                T7 RNA polymerase is recognized by poly-                   ing structurally constrained peptides and
• HSV•Tag (11 aa)                                                               clonal antibody bound to the polymerase,
    In each case, display was verified by                                       but we have not yet been able to establish                 Displayed peptides and proteins do not
either a binding assay or an enzymatic assay.                                   enzymatic activity for this phage.
                                                                                                                                           need to be capable of export through
Phage displaying T7 endonuclease appear to
                                                                                Biopanning selection                                       the periplasm and the cell membrane,
have about the same enzymatic activity as
purified T7 endonuclease (12) (Fig. 5). On                                         We have carried out standard biopan-
                                                                                                                                           as in filamentous systems.
the other hand, the activity of β-gal phage                                     ning experiments with phage displaying
could be easily detected using a standard                                       the S•Tag in high copy number or the
enzymatic assay, but was about 250-fold                                         HSV•Tag in either low or high copy num-                    peptides whose biological activity requires
lower than the measured copy number of                                          ber. S•Tag phage yielded a nearly 106-fold                 longer stretches of amino acids.
the β-gal. This difference presumably re-                                       enrichment after two rounds, and > 107-fold                   T7 phage with capsids made entirely of
flects the fact that β-gal is active only as a                                  enrichment after four rounds of biopanning                 10B grow poorly, and this may also occur
tetramer. Clearly, not all displayed enzymes                                    and growth, when the phage was originally                  with some T7Select415 phage. We know
will be active “phagezymes.” This will                                          mixed with control phage in a ratio of 1 to                that even some small peptides cannot be
depend on whether the enzyme can be                                             2 × 107 (Fig. 6). The HSV•Tag phage gave                   cloned in T7Select415, although the rules
active with a phage fused to its N-terminus                                     similar results. When the HSV•Tag was                      for exclusion are not yet understood.

                                                                                                                                                                         inNovations 6
 4
Exclusion because of either the size or the
sequence of the peptide presumably occurs
                                                                                                      Endo                   T7Select1-2/Endo T7Select1-2
because the resulting 10B/peptide fusion                                         1     2     3    4     5 6        7     8     9 10 11 12 13 14 15 16                      17
protein cannot assemble into a capsid shell.
T7Select®415 phage are normally grown on
the E. coli host BL21, where the fusion
                                                                                                                                                                                ← T7 DNA
protein is the only source of capsid protein.
In some cases, growth inhibition can be
relieved by growing the phage on BLT5403,                                                                                                                                       ← M13 DNA
where large amounts of 10A capsid protein
is provided from a plasmid. The capsid shell
of these phage will be composed of a
mixture of 10A and 10B/peptide fusion
protein.
    The largest protein yet displayed from
the low copy display T7Select1 vectors is
1015 amino acids. The primary limitation
on size is the DNA cloning capacity of the                          Fig. 5. The enzymatic activity of phage displaying T7 endonuclease.
                                                                    T7Select1-2 phage displaying T7 endonuclease and purified T7 endonuclease (12) show similar patterns of digestion of the M13
vector (3.6kbp, 1200 aa for T7Select1-1;                            ssDNA substrate. The phagezyme retained essentially the same infectivity before and after the endonuclease assay. Control phage that
2.7kbp, 900 aa for T7Select1-2 vectors).                            do not display any enzyme retained infectivity but had no effect on the M13 DNA. The band marked T7 DNA represents the T7 DNA in
                                                                    the phage particle that has been released by EDTA-heat treatment prior to loading the sample on the agarose gel. The characteristic
However, it may be that phage displaying                            smear of endonuclease digestion products is more evident in a longer exposure of the gel. T7 endonuclease (0.4 ng) and phage dis-
                                                                    playing a similar molar amount of endonuclease (assuming 0.5 copies endonuclease per phage) were used to treat M13 ssDNA (1.5
proteins of more than about 600 amino                               µg in 55 µl endonuclease buffer). 10 µl samples were taken out at 5,10, 20, 30, and 60 min after incubation at 37 °C, titered, and then
acids often grow poorly. This rule of thumb                         heated at 70 °C for 5 min in EDTA buffer prior to loading on the 1% agarose gel. Lane 1, HpaI digest of T7 DNA; lane 2, untreated
                                                                    M13 ssDNA; lanes 3–7, purified T7 endonuclease; lanes 8–12, purified T7 endonuclease phagezyme; lanes 13–17, purified control
is at least consistent with our observations                        phage.
of the behavior of phage displaying a variety
of proteins, including a series of N-terminal
                                                                   fragments of β-galactosidase (71, 271, 431,                            Summary
         1   2   3 4 5 6          7 8     9 10 11 12               691, 833 or 1015 aa). T7Select1 phage that                                The T7Select Phage Display System uses
                                                                   grow poorly can accumulate deletions                                   bacteriophage T7 as a display vector, and
                                                                   within the capsid fusion gene, since they are                          has a number of attractive features:
                                                                   grown on a complementing host (BLT5403)
                                                                                                                                          • T7 is easy to grow
                                                                   that provides the 10A protein from a plas-
                                                                   mid. The 10A gene in the plasmid is con-                               • Large display libraries can be made using
                                                                   trolled by a T7 promoter. In some cases, we                              the T7Select packaging extracts
                                                                   have relieved growth inhibition by growing                             • Peptides up to 50 aa can be displayed in
                                                                   the phage on BLT5615, where plasmid                                      high copy number (415 per phage)
                                                                   expression of 10A is controlled by the                                 • Peptides and larger proteins up to 1200
                                                          – 373
 363 –                                                             lacUV5 promoter.                                                         amino acids can be displayed in low copy
                                                          – 353
                                                                       The copy number of display from                                      number (0.1–1 per phage)
                                                                   T7Select415 grown on BL21 is fixed by the
                                                                                                                                          • Displayed peptides and proteins do not
                                                                   number of capsid proteins in the T7 capsid
                                                                                                                                            need to be capable of export through the
                                                                   shell (415). The display number from
                                                                                                                                            periplasm and the cell membrane, as in
                                                                   T7Select1 vectors is not similarly fixed. It
 Fig. 4. Cleavage of thrombin sites                                                                                                         filamentous systems
 displayed in high copy number.                                    presumably depends on the ratio of expres-
 Sufficient levels of thrombin cleaved most or all of the throm-   sion of the capsid fusion protein from the                             • T7 is extremely stable, expanding the
 bin sites (415 per phage) displayed from T7Select415, reduc-
                                                                   vector and the 10A protein from the com-                                 variety of agents that can be used in
 ing the size of the capsid protein in the shell from 373 to 353
 aa. The infectivity of the phage was not affected by thrombin     plementing host (BLT5403 or BLT5615),                                    biopanning
 treatment. Thrombin had no effect on the capsid protein (363
 aa) of the control phage. Each lane of the SDS-polyacrylamide     and also on the efficiency of assembly of the                             The capacity of phage display systems
 gel shows the proteins present in the phage particle after        fusion protein into the capsid shell. Copy                             will certainly overlap, and each will have its
 thrombin treatment. In each case, 10 ml purified phage
 (A 260 =12) was treated with thrombin for 1 hour at               numbers per phage measured by Western                                  own limitations that depend on the growth
 37 °C, titered, and then disrupted by boiling prior to loading    analysis have been 0.5 for HSV•Tag (Fig.                               properties of the phage. We have given the
 on the gel. Lanes 1–6, control phage; lanes 7–12, thrombin
 site phage. The first 5 samples in each set represent 5-fold      7), 0.3 for T7 ssDNA binding protein, 0.2                              same attention to detail and quality con-
 dilutions of thrombin from 0.5 to 0.0008 units (Novagen, cat-
 alog # 69671-1), and the sixth sample had no thrombin.
                                                                   for β-galactosidase, and 0.1 for T7 RNA                                trol with the T7Select system as with the
 These experiments were carried out by Cathryn McNamara—           polymerase.                                                                                       continued on page 6
 Brookhaven National Lab.


inNovations 6
                                                                                                                                                                                                        5
 FEATURE ARTICLE
continued from page 5
                                                                                                                                   References
                      Enrichment                                            T7Select415-1                   T7Select1-2            1. Smith, G.P. and Scott, J.K. (1993)
                                                                          1    2      3             4        5       6                 Methods in Enzymology, 217, 228–257.
                                                                                                                                   2. O'Neil, K.T. and Hoess, R.H. (1995)
                                                                                                                                       Current Opinion in Structural Biology, 5,
                                                                                                                                       443–449.
                                                                                                                                   3. Dunn, J.J. and Studier, F.W. (1983)
                                                                                                                                       J. Mol. Biol. 166, 477–535.
                                                                                                                                   4. Steven, A.C. and Trus, B.L. (1986)
                                                                                                                                       Electron Microscopy of Proteins, 5, 1–35.
                                                                                                                           – 362
                                                                                                                                   5. Russel, M. (1991) Mol. Microbiol. 5,
                    ROUND 1: 8 × 103-fold                                                                                              1607–1613.
                                                                                                                                   6. Son, M., Hayes, S.J., and Serwer, P.
                                                                                                                                       (1988) Virology, 162, 38–46.
                                                                                                                                   7. Cerritelli, M.E. and Studier, F.W.
                                                                    Fig. 7. Copy number of the HSV•Tag                                 (1996) J. Mol. Biol. 258, 286–298.
                                                                    sequence displayed from T7Select vectors.                      8. Condron, B.G., Atkins, J.F., and
                                                                    There were about 0.5 copies of HSV•Tag per T7Select1-2             Gesteland, R.F. (1991) J. Bacteriol. 173,
                                                                    phage. Phage particle proteins were analyzed by Western blot       6998–7003.
                                                                    using mouse monoclonal antibody to the epitope (Novagen,
                                                                    catalog # 69171-1). The first lane for each phage represents   9. Rosenberg, A.H. and Studier, F.W.,
                                                                    the equivalent of 25 µl of phage (A260=6), and the following       unpublished observations.
                                                                    lanes each have 10-fold less material. Lanes 1–4,
                                                                    T7Select415 displaying the HSV•Tag; lanes 5 and 6,             10. Schmidt, T.G. and Skerra, A. (1993)
                   ROUND 2: 8 × 105-fold                            T7Select1-2 displaying the HSV•Tag.                                Protein Eng. 6, 109–122.
                                                                                                                                   11. Bai, M., Harfe, B., and Freimuth, P.
                                                                                                                                       (1993) J. Virol. 67, 5198–5205.
                                                                   pET expression system, and believe that
                                                                                                                                   12. de Massy, B., Weisberg, R.A., and
                                                                   T7Select® is the system of choice for phage                         Studier, F.W. (1987) J. Mol. Biol. 193,
                                                                   display.                                                            359–376.
                                                                   Acknowledgments
                                                                      We thank Jutta Paparelli for technical
                                                                   assistance. This work was supported by a
                                                                   Cooperative Research and Development
                   ROUND 3: 7.8 × 106-fold                         Agreement (CRADA BNL-C-92-06)
                                                                   between Associated Universities, Inc. and
                                                                   Novagen, Inc., and by the Office of Health
                                                                   and Environmental Research of the United
                                                                   States Department of Energy.




                    ROUND 4: 1.5 × 107-fold



 Fig. 6. Biopanning selection of T7Select415
 phage displaying the S•Tag sequence.
 The S•Tag phage was enriched approximately 106-fold after
 two rounds of selection, and almost all of the phage con-
 tained the S•Tag after four rounds of selection. The experi-
 ments were performed starting with a mixture of phage lysate
 containing S•Tag phage and control phage in a ratio of
 1:2×10 7 (1×10 9 total infective phage). The phage were
 allowed to bind to S-protein coating a microtiter well (96-well
 plates) for 30 min at room temperature. Unbound phage were
 removed by washing with 1X PBST (phosphate-buffered
 saline, 0.1% Tween-20). Bound phage were eluted with PBST
 containing 1% SDS and used to grow a new lysate by infect-
 ing a mid-log BL21 culture. Four rounds of selection were                                                                         * The T7Select® System is covered by US patents 5,223,409,
 performed, and the identity of the phage in the lysate after                                                                        5,403,484 and other patents pending. The system is sold for re-
 each round was detected by plaque lifts followed by chemilu-                                                                        search use only. Any commercial use of the T7Select System,
 minescent visualization using the T7Select Biopanning Kit.                                                                          including the discovery or development of commercial prod-
                                                                                                                                     ucts, requires licenses from Dyax Corp. and Novagen, Inc.

                                                                                                                                                                              inNovations 6
 6
 ARTICLE

Perfectly Blunt® Cloning: A superior method
for cloning PCR products or any DNA
Robert E. Novy, Keith W. Yaeger, and Kristin M. Kolb — Novagen, Inc.




T
            he initial cloning of an unchar-                                                       excessive PCR cycles in the absence of suffi-   pends not only on the 3'– base, but also on
            acterized sequence can be ac-                                                          cient Taq DNA polymerase can lead to con-       adjacent bases. In this report, the efficiency
            celerated with use of an effi-                                                         ditions in which ragged ends are produced       of 3'–dA addition varied from 4%–75%, de-
            cient universal cloning vector.                                                        due to incomplete elongation.                   pending on the nucleotide composition at
Linearized plasmid vectors containing single                                                           Recent reports, however, indicate that      the 3'–end of the PCR product. Similar
dT overhangs (T-vectors) were originally                                                           the primary factor affecting T-vector           variations in the efficiency of 3'–dA addi-
developed as PCR product cloning vehicles                                                          cloning efficiency is the variability of 3'–nu- tion were reported by Magnuson, et al. (4).
in response to a report describing the prefer-                                                     cleotide addition mediated by Taq DNA           Both groups were able to identify cycling
ential addition of a single 3'–dA overhang                                                         polymerase.        In                                                   conditions         and
to a blunt dsDNA template when incubated                                                           addition to adding                                                      3'–base composition
with Taq DNA polymerase and dNTPs (1).                                                             dA as a 3'–over-
                                                                                                                            PCR products amplified by Taq DNA              that would generally
Although T-vector based methods have                                                               hang, Taq polymer-       polymerase exist as a heterogeneous            favor or discourage
emerged as a popular means for cloning                                                             ase is also capable mixture of ragged ends, blunt ends                  3'–dA addition. To
PCR products, many researchers have ob-                                                            of adding 3'–dC, and 3'–overhangs with the degree of                    promote 3'–dA ad-
served variable cloning efficiencies when                                                          –dG or –dT (2). The heterogeneity strongly influenced by                dition, an extended
using this strategy.                                                                               frequency of addi- nucleotides immediately flanking the                 post-PCR incuba-
    One factor known to affect T-vector                                                            tion appears to be 3'–end.                                              tion was required
cloning efficiency is the number of PCR                                                            dependent on the                                                        and the base compo-
cycles performed to produce the insert. The                                                        nucleotide occupy-                                                      sition at the 3'–ends
amount of active Taq DNA polymerase                                                                ing the 3' position of the dsDNA template.      of PCR products was restricted. These stud-
decreases with each PCR cycle, whereas the                                                         Brownstein, et al. (3) provided further proof   ies, therefore, indicate that PCR products
amount of template increases. Performing                                                           that the efficiency of 3'–dA addition de-                                  continued on page 8


          1A. Effect of insert 3'–terminus on cloning efficiency                                                                      1B. Effect of insert size on cloning efficiency

                             3'–AA              3'–TT               3'–CC               3'–GG              no insert                                   1000bp      750bp          500bp       300bp         212bp         150bp      no insert
                                                                                                                                                       T-Vector



                                                                                                                                                                    T-Vector



                                                                                                                                                                                  T-Vector



                                                                                                                                                                                               T-Vector



                                                                                                                                                                                                             T-Vector



                                                                                                                                                                                                                          T-Vector




                                                                                                                                                                                                                                      T-Vector
                                  T-Vector




                                                      T-Vector




                                                                           T-Vector




                                                                                                T-Vector




                                                                                                                   T-Vector




                                                                                                                                                       Blunt



                                                                                                                                                                    Blunt



                                                                                                                                                                                  Blunt



                                                                                                                                                                                               Blunt



                                                                                                                                                                                                             Blunt



                                                                                                                                                                                                                          Blunt




                                                                                                                                                                                                                                      Blunt
                          Blunt




                                              Blunt




                                                                   Blunt




                                                                                        Blunt




                                                                                                           Blunt




                 1800                                                                                                                         1400

                 1600                                                                                                                         1200
                                                       white colonies                                                                                                    white colonies
                 1400                                  blue colonies                                                                                                     blue colonies
                                                                                                                                              1000
                 1200
                                                                                                                              Number of cfu
 Number of cfu




                                                                                                                                               800
                 1000

                  800                                                                                                                          600

                  600
                                                                                                                                               400
                  400
                                                                                                                                               200
                  200

                    0                                                                                                                            0
                        3.3-fold*            2.7-fold            17.8-fold            24.4-fold                                                      9-fold*      4.5-fold      5.7-fold     7.3-fold     14.2-fold     12.3-fold
 Fig. 1. Blunt vs T-vector cloning efficiencies.
 1A. Four 860 bp PCR products were designed with identical sequences, except at their 3'–termini, as indicated at the top of figure 1A. The molar ratio of insert to vector for the Blunt vector and T-vector liga-
 tions was 2:1 and 5:1, respectively. These molar ratios were previously determined to be optimal for this set of inserts (data not shown). The ligation reaction was transformed into NovaBlue Competent Cells
 (efficiency ≥ 4 × 108 cfu/mg) and 0.0625 ng of vector plated in each case.
 1B. The molar ratio of insert to vector was 2.5:1 and. 0.0625 ng of vector was plated in each case.
 *Number represents the fold difference between white colonies obtained by the blunt cloning method vs. the T-vector cloning method.


InNovations 6
                                                                                                                                                                                                                                             7
 ARTICLE
continued from page 7
amplified by Taq DNA polymerase exist as a                        Colony source                                          No. with insert/                % Positive
heterogeneous mixture of ragged ends, blunt                                                                               no. analyzed

ends and 3'–overhangs with the degree of        A. pT7Blue Blunt Vector + 3'–AA 860 bp insert                                 10/10                         100%

heterogeneity being strongly influenced by          pT7Blue Blunt Vector + 3'–TT 860 bp insert                                10/10                         100%

nucleotides immediately flanking the                pT7Blue Blunt Vector + 3'–CC 860 bp insert                                10/10                         100%
3'–end. This heterogeneity is most likely re-       pT7Blue Blunt Vector + 3'–GG 860 bp insert                                10/10                         100%
sponsible for the variability observed when     B. pT7Blue Blunt Vector + 212 bp insert                                       25/25                         100%
cloning different inserts into T-vectors.
                                                Table 1. Colony PCR analysis of recombinants.
                                                White colonies were chosen at random and subjected to colony PCR with the T7 promoter primer and U19mer primer.
Avoid PCR cloning pitfalls
    Novagen has developed a streamlined
cloning procedure and kit components to         in preparation for cloning into a blunt,                            • Quickly precipitate the PCR product
avoid the pitfalls associated with cloning      dephosphorylated vector. The procedure has                            with Pellet Paint Co-Precipitant (5)
PCR products. PCR is carried out using          the following characteristics:                                        to remove dNTPs and PCR primers.
DNA polymerases that either lack 3'→5'          • Exhibits superior PCR product cloning                               Resuspend in TE buffer.
proofreading activity (Taq, Tth) or possess       efficiencies relative to T-vectors                                • Use a spin column to remove dNTPs
this activity (Pfu, DeepVent). The former                                                                             and PCR primers.
                                                • Avoids the need to limit primer
type of polymerase generates (some) prod-
                                                  composition                                                       • Isolate the desired PCR band from an
ucts with 3'–dA overhangs, while the latter
                                                • Avoids extensive post-PCR incubations                               agarose gel.
type generates products with blunt ends.
Novagen has designed the Perfectly Blunt®       • Avoids the addition of extra sequences                               With thermostable polymerases that lack
kits to facilitate the rapid cloning of PCR       to PCR primers                                                    3'→5' proofreading activity, add the PCR
products regardless of the type of polymerase   • Avoids exposure of the PCR product                                product from step 1 or 2 to a 40 minute
used for amplification. The key to this pro-      to restriction enzyme digestion                                   polish/kinase reaction, and then heat-
cedure is an optimized pretreatment step                                                                            inactivate for 10 minutes.
                                                • Allows cloning of PCR products
in which DNA ends are simultaneously                                                                                   With thermostable polymerases that pos-
                                                  amplified by any DNA polymerase
made blunt and 5'–ends are phosphorylated                                                                           sess 3'→5' proofreading activity, add the
                                                • Is as easy to use as T-vector cloning                             PCR product to a 5 minute polish/kinase
                                                  procedures                                                        reaction, and then heat-inactivate for 10
              Perfectly Blunt
                                                    With the pT7Blue and pT7Blue-2                                  minutes.
            cloning procedure
                                                Perfectly Blunt Vector kits, heterogeneous                             Add the pT7Blue or pT7Blue-2 Blunt
     1              PCR reaction                PCR product termini (or DNA with any                                Vector and ligase directly to the heat in-
                                                type of end) are converted into blunt,                              activated polish/kinase reaction and incu-
                           w                    phosphorylated ends in an optimized                                 bate at 22 °C for 1–2 hours.
                                                polish/kinase reaction. Following a brief                              Transform the ligation reaction into
     2         chloroform extraction            heat inactivation step, the blunt phosphory-                        NovaBlue competent cells and visually
                         1 minute
                                                lated insert is combined with the ready-to-                         screen for recombinants by plating in the
                           w                    use vector and ligated. Subsequent transfor-                        presence of X-gal/IPTG.
     3    PCR product cleanup (optional)
                                                mation into the highly efficient NovaBlue
                 Pellet Paint: 10 minutes       Competent Cells generates recombinant                               Comparison of blunt vectors and
                  gel extraction: 2 hours
                            w                   colonies that are visualized easily by                              T-vectors
                                                blue/white screening. The vectors are shown
                                                                                                                       We compared the blunt vector and
     4         polish/kinase reaction           schematically in Figure 4. A summarized
                  Taq, etc.: 40 minutes                                                                             T-vector cloning methods in two sets of ex-
                   Pfu, etc.: 5 minutes         version of the Perfectly Blunt cloning pro-
                                                                                                                    periments. In each case, the PCR products
                            w                   cedure is outlined in the box to the left.
                                                                                                                    were generated using Taq DNA polymerase,
     5           heat inactivation                                                                                  1.5 mM MgCl2, and the following cycling
                        10 minutes              Blunt vector cloning procedure
                                                                                                                    profile; 94 °C for 1 minute, 52 °C for
                           w                        After amplification, the PCR reaction is                        1 minute, 72 °C for 2 minutes with a
                                                vigorously extracted with CHCl3 to inacti-                          3 second/cycle auto extension, and a final
     6                  ligation
                        1–2 hours               vate the polymerase. One µl of the PCR                              72 °C incubation for 15 minutes.
                                                reaction may be added directly to the                                  In the first set of experiments, we tested
                           w
                                                polish/kinase reaction at this point. How-                          the cloning efficiencies of PCR products of
     7            transformation                ever, higher cloning efficiencies are ob-                           identical size that differed only by the base
                                                tained by using one of the following cleanup                        composition at their termini.
                                                procedures:

                                                                                                                                                              inNovations 6
 8
                  Vector + insert                                       No. of junctions                Sequencing results                            were cloned with high efficiency by both
                                                                           analyzed                       as expected?                                methods, the blunt vector method produced
A. pT7Blue Blunt Vector + 3'–AA 860 bp insert                                  2                                     yes                              approximately 3-fold more positives.
    pT7Blue Blunt Vector + 3'–TT 860 bp insert                                 2                                     yes                              Remarkably, when the inserts contained
    pT7Blue Blunt Vector + 3'–CC 860 bp insert                                 2                                     yes                              3'–CC or 3'–GG termini the blunt vector
    pT7Blue Blunt Vector + 3'–GG 860 bp insert                                 2                                     yes                              method was substantially more efficient,
B. pT7Blue Blunt Vector + 212 bp insert                                        8                                     yes                              yielding 18-fold and 24-fold more positives,
                                                                                                                                                      respectively. Moreover, significantly greater
Table 2. Sequence analysis of vector-insert junctions.
Cycle sequencing was performed with Cy5-labeled primers and a Pharmacia ALF Express sequencer.                                                        overall cloning consistency was observed
                                                                                                                                                      in the blunt vector method. The variation
                                                                                                                                                      between the most efficiently cloned insert
   Four 860 base pair (bp) PCR products                            tained for each vector-insert combination                                          and the least efficiently cloned insert was
which had the following sequences at their                         are illustrated in Figure 1, parts A and B.                                        only 2.5-fold for the blunt vector, but was
termini were used:                                                     As shown in Figure 1, both cloning                                             12.8-fold for the T-vector. Similar results
                                                                   methods gave good signal-to-noise ratios                                           were obtained when the four 860 bp inserts
3'–AA/860 bp insert :
5'–TTTCATGA----- //----- TCATGAAA–3'                               (number of colony forming units [cfu]                                              were cloned into another commercially
3'–AAAGTACT----- //----- AGTACTTT–5'                               obtained + insert ÷ number of cfu obtained                                         available T-vector (data not shown).
3'–TT/860 bp insert :                                              in the absence of insert). However, the                                                The data in Figure 1B further illustrate
5'–AATCATGA----- //----- TCATGATT–3'                               signal-to-noise ratio observed with the                                            the superior efficiency of the blunt vector
3'–TTAGTACT----- //----- AGTACTAA–5'                               blunt vector was consistently better than                                          method. Six inserts of varying size and
3'–CC/860 bp insert :                                              that obtained from the T-vector method.                                            3'–end composition were cloned by each
5'–GGTCATGA----- //----- TCATGACC–3'                                   Figure 1A also reveals the dramatic                                            method. The blunt vector method yielded
3'–CCAGTACT----- //----- AGTACTGG–5'
                                                                   effect that 3'–base composition can have on                                        cloning efficiencies that were 4.5-fold to
3'–GG/860 bp insert :                                              relative cloning efficiency with a blunt                                           14-fold higher than those obtained with the
5'–CCTCATGA----- //----- TCATGAGG–3'
3'–GGAGTACT----- //----- AGTACTCC–5'                               vector vs. a T-vector. Although the 860 bp                                         T-vector method.
                                                                   inserts with the 3'–AA or 3'–TT termini                                                                     continued on page 10
Note that for each respective PCR product,
the last eight bases at each 3'–end of the
DNA are identical. Also note that only the                            2A.
                                                                                             kb     1    2 3         4     5 6     7 8 9 10 11 12 13 14 15
first two and the last two base pair positions
vary between the four PCR products. The
PCR products were designed in this way
                                                                                         2000 –
to directly measure the effect of 3'–base                                                1500 –
                                                                                         1000 –
composition on blunt vector and T-vector                                                  750 –
                                                                                          500 –
cloning efficiency.                                                                       300 –
                                                                                          150 –
    In the second set of experiments, we                                                   50 –

tested the cloning efficiencies of PCR prod-
                                                                                                            1         Novagen’s PCR Markers
ucts having various sizes (1000 bp, 750 bp,                                                                 2, 3      no template
                                                                                                            4, 5      uncut pT7Blue template (positive control)
500 bp, 300 bp, 212 bp, 150 bp) and with
                                                                                                            6 – 15    random white colonies from the blunt vector
different 3'–end composition.                                         2B.                                             + 3'–CC 860 bp insert reaction


Generation of recombinant colonies                                                            kb
                                                                                                    1   2    3 4      5    6 7 8      9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

   The PCR products described above                                                        2000 –
                                                                                           1500 –
were purified from agarose gel slices using                                                1000 –
                                                                                            750 –
QIAEx II (Qiagen, Inc.) and eluted in TE.                                                  500 –
                                                                                            300 –
After quantitation by absorbance at 260                                                     150 –
                                                                                             50 –
nm, the PCR products were ligated to
                                                                                                                                 1        Novagen’s PCR Markers
pT7Blue T-vector or processed through                                                                                            2        no template
the polish/kinase reaction prior to ligation                                                                                     3        uncut pT7Blue template (positive control)
                                                                                                                                 4 – 28   random white colonies from the blunt vector
to pT7Blue Blunt Vector. One microliter                                                                                                   + 212 bp insert reaction
of each ligation was transformed into
NovaBlue Competent Cells. Samples of the
                                                                      Fig. 2. Colony PCR gel results.
transformation mixture containing identical
                                                                      2A. Colony PCR analysis of random white colonies from the pT7Blue Blunt Vector + 3'-CC/860 bp insert plate.
amounts of vector were plated (0.0625 ng).                            2B. Colony PCR analysis of random white colonies from the pT7Blue Blunt Vector + 212 bp insert.
The numbers of blue and white colonies ob-

InNovations 6
                                                                                                                                                                                                   9
 ARTICLE
continued from page 9
                                                                  insert:vector recommended for the blunt          frame with the ORF of the pT7Blue-2 Blunt
                   1       2      3       4
                                                                  vector method (1:1 to 2.5:1), the observed       Vector, when cloned in the proper orien-
                                                                  frequency of multimer formation is ≤ 10%.        tation. The combined vector-insert ORF
                                                                  Although unexpected, dimers are also ob-         would generate a 312 amino acid polypep-
                                                                  served when cloning into T-vectors. In this      tide (73 aa from the vector + 239 aa from
                                                                  case, dimers are presumably obtained by          the insert).
                                                                  the ligation of two PCR products that                When a sample of the transformation
                                                                  both possess one blunt end and one 3'–dA         mixture containing 0.0625 ng of vector
                                                                  extended end.                                    from the ligation reaction was plated, 478
                                                                                                                   white colonies were obtained. We then used
 Fig. 3. Transcription/translation analysis                       DNA sequence analysis of
 with pT7Blue-2 Blunt Vector.                                                                                      the R-20mer upstream primer and a
                                                                  recombinants
 The 4 bp 3' and 5' overhangs of a 753 bp DNA fragment were                                                        3'–insert specific primer to PCR-amplify
 converted to blunt ends in a polish/kinase reaction. The
 resulting DNA fragment encoding a 239 amino acid ORF was
                                                                      Colony PCR positives were next sub-          several colonies. The same primer combina-
 cloned into pT7Blue-2 Blunt Vector and transformed. The          jected to DNA sequence analysis to exam-         tion was also used to amplify the original
 original ligation and the white colony transformants were
 amplified with the R-20mer primer (Novagen catalog # 69835-      ine the vector-insert cloning junctions. Two     blunt vector + insert ligation reaction ac-
 1) and an insert-specific primer. This yielded a PCR product     junctions from each of the pT7Blue Blunt         cording to Novagen’s Primers-to-Protein
 containing a T7 promoter, 5' UTR globin translation enhancer
 and a 312 amino acid vector-insert ORF. The PCR product          Vector + 860 bp recombinants and 8 junc-         protocol. These PCR products served as
 was then transcribed and translated. 35S-met labeled transla-    tions from the 212 bp recombinants were se-
 tion products were analyzed by SDS-PAGE followed by fluo-
                                                                                                                   input DNA for in vitro transcription/transla-
 rography. Lane 1, pT7Blue-2 gus positive control template;       quenced. As shown in Table 2, all sixteen of     tion analysis. 35S-Met was included in the
 lane 2, pT7Blue-2 β-gal positive control template; lanes 3, 4,   the junctions analyzed contained the ex-
 pT7Blue-2 Kpn I/EcoR I insert translation product obtained                                                        translation reactions to label the synthesized
 from ligation PCR and colony PCR DNA templates, respec-          pected vector-insert sequence. This result       protein and a fluorograph of the translation
 tively.
                                                                  demonstrates the high fidelity of blunt end      products is shown in Figure 3.
                                                                  formation during the combined polish/                Both PCR reactions yielded translation
                                                                  kinase reaction.                                 products of the expected size, indicating
PCR analysis of recombinants
                                                                      All results described here verify that the   that the appropriate vector-insert junctions
    Colony PCR analysis was performed to                          pT7Blue Perfectly Blunt kit significantly en-    had been formed in the blunt vector liga-
verify that the white colonies in Figure 1A                       hances overall PCR product cloning effi-         tion. Sequence analysis confirmed the cre-
and 1B contained inserts. Table 1A shows                          ciency while significantly decreasing the        ation of the expected vector-insert junction
the results obtained when 10 colonies from                        variability in cloning efficiency attributable   (data not shown).
each pT7Blue Blunt Vector + 860 bp insert                         to 3'–termini. Similar results were also ob-
plate were analyzed.                                              tained with the pT7Blue-2 Perfectly Blunt        Conclusions
    In every case, all 10 of the colonies con-                    kit using the same PCR products described            Data presented here show that the
tained inserts. Figure 2A shows agarose gel                       above (data not shown). The pT7Blue-2            pT7Blue and pT7Blue-2 Perfectly Blunt
analysis of PCR products obtained from the                        Blunt Vector is a blue/white screening vec-      cloning kits provide better consistency and
10 colonies containing the pT7Blue Blunt                          tor designed to serve as a general cloning       higher efficiency than vectors containing
Vector + 3'–CC/860 bp insert. Nine of the                         vehicle and as a vector for optimal in vitro     single 3'–dT overhangs for cloning PCR
ten colonies analyzed in Figure 2A yielded                        transcription/translation (see Fig. 4).          products generated by Taq DNA poly-
the expected 999 bp. The other colony                                                                              merase. This result may be somewhat
yielded a larger PCR product whose size was                       Cloning inserts possessing                       surprising, since it is expected that even a
consistent with the presence of two tandem                        5' and 3' overhangs                              single base overhang would provide an ad-
inserts.                                                              To demonstrate the general utility of        vantage over blunt-end ligation. However,
    Table 1B and Figure 2B show the analo-                        pT7Blue-2 and the blunt vector cloning           our data clearly show a strong dependence
gous data obtained when colonies were                             method, a 753 bp DNA fragment with an            of T-vector cloning efficiency on the termi-
examined for the presence of the 212 bp                           open reading frame of 239 amino acids was        nal bases of the PCR products. It would thus
insert. In this case, a colony containing a                       generated by restriction enzyme digest. The      appear that under commonly used PCR con-
212 bp insert should yield a 351 bp PCR                           restriction enzymes left a 4 bp 3'–overhang      ditions, a high degree of end heterogeneity
product. All 25 colonies analyzed contained                       at one end (Kpn I) and a 4 bp 5'–overhang        must exist, which results in a relatively low
an insert, and 2 contained an insert dimer.                       (EcoR I) at the other end. The overhangs         proportion of molecules containing single
Note that because the blunt vector method                         were converted to blunt ends in the              3'–dA overhangs on both ends. Because this
produces inserts that may ligate with each                        polish/kinase reaction and the insert was lig-   system uses blunt-end ligation of insert to
other, a low percentage of recombinants                           ated to the pT7Blue-2 Blunt Vector. The          a dephosphorylated vector, several other
containing tandem inserts is expected. The                        cloning was planned so that correct process-     advantages are also realized:
frequency of multimer formation is,                               ing of the ends (4 bp chew back on the           • Extraneous sequences, such as restriction
however, directly related to the ligation                         3'–overhang and 4 bp fill-in on the 5'–over-        sites or nucleotides that promote 3'–dA
conditions. At the low molar ratios of                            hang) would produce an insert ORF in-               addition, do not need to be added to PCR

                                                                                                                                                 inNovations 6
10
                                                                                                                                         1. Clark, J. M. (1988) Nucleic Acids Res.
  pT7Blue Blunt Vector                                                                                                                      16, 9677–9686.
                                                                                                                                         2. Hu,G. (1993) DNA and Cell Biology 12,




                                   Sse8387 I, Pst I




                                   ✸ EcoR V ✸
                                                                                                                                            763–770.




                                   BamH I
                                   Hind III




                                   EcoR I
                                   Sma I
                                   Sph I




                                   Nde I



                                   Kpn I
                                   Xba I
                                   Spe I




                                   Sac I
                                   Sal I
                                                                                                                                         3. Brownstein, J. M., Carpten, J. D. and
  R20 mer primer
                                                                                                                                            Smith, J. R. (1996) BioTechniques 20,
    lacZ ORF
                   T7 promoter
                                                    MCS                                lacZ ORF                                             1004–1010.
                                                                                                                                         4. Magnuson, V.L., Ally, P.S., Nylund,
                                                                       U19mer primer
                                                                                                                                            S.J., Karanjuwala, Z.E., Rayman, J.B.,
                                                                                                                                            Knapp, J.I., Lowe, A.L., Ghosh, S., and
                                                                                                                                            Collins, F.S. (1996) BioTechniques 21,
  pT7Blue-2 Blunt Vector                                                                                                                    700–709.
                                                                   ✸ Srf I, Sma I ✸                                                      5. McCormick, M. (1996) inNovations 5,
                                                                                                                                            10.




                                                                                                  BstX I, Pml I
                                                                                                  EcoO109 I
                                                                   BamH I




                                                                                                  Hind III
                                                                                                  EcoR V
                                                                   SnaB I

                                                                   EcoN I
                                                                   BseR I




                                                                                                  EcoR I
                                          Msc I
                                          Nco I




                                                          Kpn I




                                                                   Mlu I




                                                                                                  Xho I
                                                                                                  Avr II
                                                                                                  Nhe I
                                                          Age I




                                                                                                  Xba I


                                                                                                  Apa I
                                                                                                  Sac I
                                                                                                  Not I
                                                                   Pst I




                                                                                                  Sal I
  R20 mer primer

                                                                                        SP6 promoter
    lacZ ORF T7 promoter         globin           S•Tag           ek      MCS                          MCS                    lacZ ORF
                                 5'–UTR

                                                                                                              U19mer primer              The PCR process is covered by patents owned by Hoffmann-
                                                                                                                                         La Roche, Inc. and F. Hoffmann-La Roche Ltd.
  ✸ blunt cloning site
                                                                                                                                         DeepVent is a trademark of New England Biolabs, Inc. QIAEx II is
                                                                                                                                         a trademark of Qiagen, Inc.
 Fig. 4. Multiple cloning sites and PCR primer sites for the pT7Blue and pT7Blue-2 Blunt
 Vectors, respectively.


  primers.                                                                                niques such as restriction digests, cDNA
• The PCR product, unlike other blunt                                                     synthesis, DNA shearing, and DNase
  ligation cloning schemes, is not exposed                                                digests may be used as input DNA into
  to a restriction enzyme during the                                                      this system.
  ligation step.                                                                           Theoretically, the only disadvantage of
• PCR products produced by thermostable                                                this system is the potential for cloning mul-
  DNA polymerases such as Pfu                                                          tiple inserts. However, as seen from the
  (Stratagene Cloning Systems) and                                                     data, the frequency of cloning multiple in-
  Deep Vent (New England BioLabs,                                                     serts was less than 10%, thereby effectively
  Inc.), which possess 3'→5' proofreading                                              eliminating this as a serious concern. The
  activity (and already have blunt ends),                                              blunt vector strategy thus provides an end-
  are also compatible with the system.                                                 independent method for convenient cloning
• In addition to PCR products, DNA                                                     of any type of DNA.
  generated from commonly used tech-
                                                                                       References




InNovations 6
                                                                                                                                                                                                  11
 NEW PRODUCTS




                                                                                                                NewProducts
T7Select Phage Display System
    Novagen’s T7Select Phage Display System is         menting host (BLT5403) that supplies large
a novel display system that takes advantage of the     amounts of the 10A capsid protein from a plas-
properties of bacteriophage T7. Target peptides or     mid. Proteins up to 1200 amino acids can be dis-
proteins are expressed as fusions to the C-termi-      played from T7Select1-1b. The T7Select1-2
nus of the 10B capsid protein (near amino acid         series has a somewhat smaller capacity (900
348), and are displayed on the virion surface,         amino acids), but is available with multiple
where they are accessible for interaction with         cloning sites in all three reading frames, desig-
other proteins or ligands. Phage containing 10B        nated a, b, and c by the same convention used in
fusion proteins are infective, and a variety of bio-   the pET expression vectors.
logically active peptides and proteins have been
successfully displayed in this system.
    T7Select vectors are available for high-copy
number display of peptides or low-copy number
display of either peptides or larger proteins. The
T7Select415 vector displays 415 copies of pep-
tides up to 50 aa in size on the surface of the
capsid. Its capsid shell is composed entirely of the
fusion protein, and the phage grows on standard
E. coli hosts, such as BL21. The T7Select1 vec-
tors display either peptides or proteins in low-
copy number (0.1–1 per phage). Low-copy
number display was made possible by greatly re-
ducing expression of the capsid protein from the
vector. T7Select1 phage are grown on a comple-
  Vector                 Application           Copy Number        Cloning Capacity               Host
  T7Select415-1            Peptides                415                0–50 aa                   BL21
  T7Select1-1        Peptides or proteins          ≤1                0–1200 aa                 BLT5403
  T7Select1-2        Peptides or proteins          ≤1                 0–900 aa                 BLT5403



T7Select Cloning kits include the                      • 1010 pfu T7Select Positive Control Lysate
following components for construction of up            • 1010 pfu T7Select Negative Control Lysate
to 5 libraries:                                        • 50 µl     S-protein HRP Conjugate
• 5 µg        T7Select1-1 or T7Select415-1             • 2 ml      T7Select S-protein
              EcoR I/Hind III Vector Arms              • 2 × 25 ml SuperSignal CL-HRP
• 0.2 pmol T7Select Control Insert                                 Substrate
•6            T7 Packaging Extracts                    • 25        gLOCATOR Luminescent
• 1 µg        T7Select Packaging Control                           Labels
              DNA                                      • 25        Development Folders
• 0.2 ml      BLT5403 or BL21 Glycerol                 •1          T7Select System Manual
              Stock
• 500 pmol T7SelectUP primer
                                                       Product                          Size        Cat. #
• 500 pmol T7SelectDOWN primer
                                                       T7Select1-1 Cloning Kit                   70010-3
•1            T7Select System Manual
                                                       T7Select415-1 Cloning Kit                 70015-3
T7Select Biopanning Kit includes the fol-              T7Select Biopanning Kit                   70018-3
lowing components for biopanning with                  T7Select Packaging Kit 6 extracts         70014-3
target ligands using 96-well microplates or            Uncut T7Select415-1b DNA 10 µg            70040-3
similar solid phases:                                  Uncut T7Select1-1b DNA     10 µg          70048-3
• 25 g        Blocking Reagent                         Uncut T7Select1-2a DNA     10 µg          70042-3
• 50 ml       10X TBST Wash Buffer                     Uncut T7Select1-2b DNA     10 µg          70043-3
• 10 ml       T7 Elution Buffer                        Uncut T7Select1-2c DNA     10 µg          70044-3

• 0.2 ml      BL21 Glycerol Stock
• 0.2 ml      BLT5403 Glycerol Stock                   SuperSignal is a trademark of Pierce Chemical Company.




inNovations 6
                                                                                                                         13
 NEW PRODUCTS

Perfectly Blunt® Cloning Kits
    The Perfectly Blunt® Cloning Kits are       ates recombinant colonies that are visual-            The Perfectly Blunt Cloning Kits con-
designed for simplified cloning of any DNA       ized easily by blue/white screening. Refer to     tain enough reagents to perform 40 ligations
fragment regardless of whether the termini      the article Perfectly Blunt Cloning: A superior   and transformations:
are blunt ends, 5'–overhangs or 3'–over-        method for cloning PCR products or any DNA        • 2 µg       pT7Blue or pT7Blue-2 Blunt
hangs. DNA fragments generated from com-        (pp 7–11) for details.                                         Vector
monly used molecular biology techniques                                                           • 10 µl      Positive Control Insert
                                                   Advantages of the Perfectly Blunt
such as PCR, restriction digests, cDNA syn-     Cloning Kits include:                             • 50 µl      10X PK Buffer
thesis, DNA shearing, and DNase digests                                                           • 50 µl      PK Enzyme Mix
                                                • Inserts with any type of end can be
may be used as input DNA into this system.                                                        • 2 × 100 U T4 DNA Ligase
                                                  cloned.
The Perfectly Blunt Cloning Kits are an                                                           • 25 µl      100mM DTT
ideal choice for high efficiency cloning of     • Cloning is highly efficient.
                                                                                                  • 1.5 ml     Nuclease-free Water
PCR products because they avoid the pit-        • Use of proofreading thermostable                • 5 × 0.2 ml NovaBlue Competent Cells
falls associated with T-vector cloning.           DNA polymerases is possible.                    • 4 × 2 ml SOC Medium
    With the Perfectly Blunt Cloning Kits,      • Cloning is not dependent on 3'–dA               • 5 µl       Test Plasmid
the target DNA is first converted to a blunt,     overhangs or other nucleotide                   Product                                   Cat. #
phosphorylated form in a single, brief reac-      overhangs.                                      pT7Blue Perfectly Blunt Cloning Kit     69901-3
tion. Following a brief heat inactivation                                                         pT7Blue-2 Perfectly Blunt Cloning Kit   70071-3
                                                • No restriction enzymes are necessary.
step, the blunt phosphorylated insert is                                                          Pellet Paint Co-Precipitant             69049-3
combined with blunt dephosphorylated            • Recombinant colonies are easily visual-
                                                                                                  Single Tube Protein System 3            70192-3
pT7Blue or pT7Blue-2 vector and ligated.          ized with blue/white screening of inserts.
                                                                                                  Primers-to-Protein System 3             70208-3
Subsequent transformation into the highly                                                         U-19mer primer                          69819-1
efficient NovaBlue Competent Cells gener-                                                         R-20mer primer                          69835-1




Xa/LIC Vector Kits
    Novagen recently introduced a set of li-    cleaves on the C-terminal side of its recog-      • 50 µl      10X T4 DNA Polymerase
gation independent cloning (LIC) vectors        nition site, a protein possessing any desired                  Buffer
for the efficient directional cloning of PCR    amino-terminus can be generated by Xa             • 100 µl     100 mM DTT
products into pET, pBAC, and pT7Blue-2          cleavage.                                         • 50 µl      100 mM EDTA
vectors. The LIC site in this set of vectors        Xa/LIC Vector Kits are available for E.       • 1.5 ml     Nuclease-free Water
encodes an enterokinase cleavage site           coli expression (pET-30 Xa/LIC & pET-32           • 2 × 0.2 ml NovaBlue Competent Cells
(DDDDK). Recombinants generated from            Xa/LIC). Each kit provides sufficient             • 1 × 0.2 ml BL21(DE3) Competent Cells
these vectors encode Met or Ile immediately     reagents for 20 annealings and transforma-        • 1 × 0.2 ml BL21(DE3)pLysS Competent
after the enterokinase site. Our LIC reper-     tions. A Control Insert is included to verify                  Cells
toire has expanded to include two new vec-      performance.                                      • 2 × 2 ml SOC Medium
tors, pET-30Xa/LIC and pET-32Xa/LIC,            Components:                                       • 5 µl       Test Plasmid
which encode a cleavage site for Factor Xa      • 1 µg      pET-30 or pET-32 Xa/LIC               Product                                   Cat. #
(IEGR). The Xa/LIC site is designed to                      Vector                                pET-32 Xa/LIC Vector Kit                70072-3
allow the directional cloning of a DNA          • 0.4 µg    Xa/LIC Control Insert                 pET-30 Xa/LIC Vector Kit                70073-3
insert encoding any amino acid directly         • 25 U      T4 DNA Polymerase
after the Xa site (IEGR). Because Factor Xa




                                                                                                                                          inNovations 6
14

				
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