OUTLINE Expression and manipulation of transgenes in the laboratory by kaitlynnbarton

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                                      OUTLINE

       Expression and manipulation of transgenes in the laboratory
To study the protein: Express your transgene
     Usually in E. coli, for speed, economy
     Expression in eukaryotic hosts
     Drive it with a promoter/enhancer
     Purify it via a protein tag
     Cleave it to get the pure protein

In vitro mutagenesis to isolate variants of your protein or gene with desirable properties
      Single base mutations
      Deletions
      Cassette mutagenesis

Explore protein-protein interaction
    Co-immunoprecipitation (co-IP) from extracts
    2-hybrid formation
    surface plasmon resonance
    FRET (Fluorescence resonance energy transfer)
    Complementation readout



                                                                                             Class 15
                                 2
Promoters for E. coli




                        ;




                            Class 15
                                                                                                                      3
                    Inducible expression in E. coli: T7 system

Component                                           -IPTG                                   +IPTG

Lac repressor gene (I-gene)                                     constitutively on
T7 RNA Polymerase gene driven by the lac promoter     off                                   on
Your favorite gene driven by the T7 promoter          off                                   ON

                                              Two-stage amplification of induction:
                                              Many T7 Pol molecules from one T7 pol gene
        plasmid                               Many YFG molecules for every T7pol molecule
                                              Does not need to compete with
                                              E. coli RNA pol; all the T7 pol can be used for the transgene transcription.

 T7 prom.   YFG



                                                                                                    chromosome
                                                       Lac promoter       T7 RNA
                                                                         polymerase

                                                      +IPTG



               Constitutive synthesis
                                                                                                    chromosome
                                                      Lac promoter       T7 RNA
         Lac I (repressor)
                                                                        polymerase

                                                                                                                 Class 15
His tag allows easy purification of the cloned gene product
                                                                                     4




                              (Nitriloacetic acid
                               chelate, NTA)



                                                          (competes with his)




                                                    Other cell proteins




                                                                                Class 15
                                                                                                                 5
                       Assorted protein tags used for purification

                                                                                                           Could be
   (IgG-binding domain from Protein A)                                                                     harmful



             (binds to biotin site)

            (Gets biotinylated in vivo)

                                                                      (poly-glucose)

                    (Amp-resistance)
                                                                          (tripeptide incl cys)




Protein A is a bacterial protein that binds tightly and specifically to IgG
Avidin from egg white binds biotin strongly (Kd~10-15).
Streptavidin similar, from bacteria, cleaner (less charged, not glycosylated)
Proteins can be biotinylated in vivo by bacterial enzymes acting on specific peptide substrates (at lys)
MBP = maltose binding protein
GST = glutathione-S-transferase


                                          E-Xaa-Xaa-Y -Xaa-Q-(G/S),


                                                                                                            Class 15
                                                                          6

Cleave a fusion protein (like GST-fusions) using TEV
(tobacco etch virus) protease:

Insert a specific substrate sequence between the 2 protein domains
(E-Xaa-Xaa-Y -Xaa-Q-(G/S): highly specific)

Add enzyme. Separate pure protein.




 Protein of interest                    Tagging protein:
                                        GFP, GST, MBP, etc.




                               +
                                                                     Class 15
                                                              7

               Tag removal after purification




                           OR

       Enterokinase:       Asp Asp Asp Asp Lys ……
                           OR

TEV protease (tobacco etch virus):   Glu-X-X-Tyr-X-Gln-Ser


                                                         Class 15
                                                                                   8
                                     E. coli as a host


•   Easy, flexible, high tech, fast, cheap;
    but problems

•   Folding (can misfold)
•   Sorting -> can form inclusion bodies
•   Purification -- endotoxins
•   Modification -- not done (glycosylation, phosphorylation, etc. )
•   Glycoproteins
•   Acylation: acetylation, myristoylation
•   Methylation (arg, lys)
•   Phosphorylation (ser, thr, tyr)
•   Sulfation (tyr)
•   Prenylation (farnesyl, geranylgeranyl on cys)
•   Vitamin C-Dependent Modifications (hydroxylation of proline and lysine)
•   Vitamin K-Dependent Modifications (gamma carboxylation of glu)
•   Selenoproteins (seleno-cys from ser tRNA at stop)




                                                                              Class 15
                                                    9

                      Some alternative hosts

•   Yeasts (Saccharomyces , Pichia)
•   Insect cells with baculovirus vectors
•   Mammalian cells in culture (later)
•   Whole organisms (mice, goats, corn)
    (not discussed)

• In vitro (cell-free), for analysis only
    (good for radiolabeled proteins)




                                               Class 15
                                                                        10
                                Yeast Expression Vector

2mm-seq = yeast ori
oriE = bacterial ori
                                             2mm plasmid
Ampr = bacterial selection
                                                           GAPDterm
LEU2 = Leu biosynthesis
         yeast selection


                                                                Gene of
                                 LEU2
                                                                interest
   Complementation of an
   auxotrophy can be used
   instead of drug-resistance
                                                                 (GOI)




 Saccharomyces cerevisiae
      (baker’s yeast)                     Ampr             GAPDprom
                                                    oriE
                                                                      Class 15
                                                                 11
             Yeast -Genomic integration

                    t                  p            Vector
                                                     DNA
                        YFG




                        HIS4
                                                    Genomic
                                                     DNA




                                                           Genomic
                               HIS4-
                                                            DNA
                p                          t
                        YFG
Functional                                     Defective
HIS4 gene                                      HIS4 gene

                                                              Class 15
                                                                                       12
   Double recombination Yeast (integration in Pichia pastoris)
P. pastoris
-tight control                              HIS4
-methanol induced (AOX1)                                        Vector
-large scale production                                          DNA
   (gram quantities)    AOX1t




                                GOI
                    AOX1p                                  3’AOX1


Genomic
 DNA
                                 Alcohol oxidase gene
                                      AOX1 gene (<= 30% of protein)
                                                                             Genomic
                                                                              DNA



                   GOI
          AOX1p             AOX1t             HIS4                       3’AOX1
                                                                                  Class 15
                                                                      13
                           Half-genes

                                           Linearized
                                           viral genome




Strong polyhedron
promoter
                                                   (Fall army worm)



                    Baculovirus as a vector
                         in insect cells
                                                                 Class 15
                                                                                       14

                                                                      Linearized
                                  Half-gene
     Constitutive early to late                                       viral genome
     promoter


Strong polyhedron promoter




                                          Re-built ORF1629,
Re-built functional lacZ (blue)
                                          required for viral growth




                 Recombination to yield
                     active virus




           Gene of interest

                                                                                     Class 15
Baculovirus cont.                                        15




                    Incubate 2-3 days




                                        Enterokinase




                                                       Class 15
                                                                          16
In vitro translation following in vitro transcription
 Transcription to RNA via the T7 promoter + T7 pol
 Rabbit Reticulocyte lysate
 Or wheat Germ Lysate
 Or E. coli lysate (combined transcription + translation)

Add ATP, GTP, tRNAs, amino acids, label (35S-met),
May need to add RNase (Ca++-dependent)
to remove endogenous mRNA

                 VECTOR

                                                    cDNA

                          T7 RNA polymerase
                          binding site (17-21 nt)
                                ACCATGG…..
                                                      Labeled protein
                                                                        Class 15
                                                                       17

   Mammalian cell expression

   Popular hosts cell lines:

   HeLa (human cevial cancer)
   HEK293 (human embryonic kidney
   Vero (monkey)
   Cos (monkey)
   Mouse 3T3
   CHO (Chinese hamster ovary)
   BHK (baby hamster kidney)


Generalized gene structure for mammalian expression:
                                                        polyA site

       Mam. prom.   intron     cDNA gene
                    5’UTR                       3’UTR




                                                                     Class 15
                                                                     18

Expression in mammalian cells

HEK293 Human embyonic kidney
HeLa   Human cervical carcinoma\
CHO    Chinese hamster ovary
Cos    Monkey cells with makingSV40 replication proteins
BHK    Baby hamster kidey
HepG2 Human hepatoma
3T3    Mouse or human exhibiting ~regulated (normal-like) growth
GH3    Rat pituitary cells
PC12   Mouse neuronal-like tumor cells
MCF7 Human breast cancer

+ various others, many differentiated to different degrees

AND:

Primary cells cultured with a limited lifetime.
E.g.,    MEF = mouse embryonic fibroblasts
         HDF = Human diploid fibroblasts


                                                                   Class 15
                                                                 19
      Expression in mammalian cells: HEK293, HeLa, CHO, cos,
                  popular mammalian cell promoters


•   SV40 LargeT Ag (Simian Virus 40)
•   RSV LTR (Rous sarcoma virus)
•   MMTV (steroid inducible) (Mouse mammary tumor virus)
•   HSV TK (low expression) (Herpes simplex virus)
•   Metallothionein (metal inducible, Cd+)
•   CMV early (Cytomegalovirus)
•   Inducible / repressible:
     tet, ecdysone, glucocorticoid (tet = tetracycline)



                                                               Class 15
                 20




Got this far




               Class 15
                    Regulated expression:                                                21
                    Tetracycline-reponsive promoters
                    Tet-OFF (add tet  shut off)
                                                   tetR   VP16 tc’n
                                                   domain act’n domain

tTA = tet activator fusion protein:
                                                                             active
                   No tet
                   Binds tet operator
                   (if tet not bound)

                                                    tetR
                                                    domain VP16 tc’n
                                                           act’n domain
                Tetracycline (tet), or,
                better, doxicyclin (dox)
                                                                          not active



                                                                polyA site

       CMV prom.                        tTA cDNA




                                                                                       Class 15
                                                                                          polyA site 22

                  MINIMUM CMV prom.              your favorite gene

Mutliple tet operator elements


                                       tetR   VP16 tc’n
                                       domain act’n domain

Tet (dox) present:                                           not active
                                                             little transcripton (bkgd)
                                                                                          polyA site

                   MINIMUM CMV prom.             your favorite gene


 Remove tet (dox):

         tetR   VP16 tc’n
         domain act’n domain
                                         active
                                         Plenty of transcripton (> 50X)                   polyA site

                  MINIMUM CMV prom.             your favorite gene

                                                                                                   Class 15
                                                                                       23
            Tetracycline-reponsive promoters
            Tet-ON (add tet  turn on gene
                                             tetR   VP16 tc’n
                                             domain act’n domain
            Diff. fusion protein:                                       not active
            Does NOT bind tet operator
            (if tet not bound)


                                             tetR   VP16 tc’n
                                             domain act’n domain

                                                                            active


                  Tetracycline (tet), or,
                  better, doxicyclin (dox)

                                                               polyA site

CMV prom.                     tTA cDNA


                                                 Binds tet operator
                                                 (if tet not bound)

                                                                                     Class 15
Tet-ON
                                                                                           polyA site 24

                   MINIMUM CMV prom.                    your favorite gene

Mutliple tet operator elements

                                        tetR   VP16 tc’n
                                        domain act’n domain

        Tet (dox) absent:                                         not active
                                                                  (little transcription)
                                                                                           polyA site

                    MINIMUM CMV prom.                   your favorite gene


 Add tet (dox):

Tet
or       tetR   VP16 tc’n                                     active
dox      domain act’n domain                                  little transcripton (bkgd)
                                                 active
                                                 Plenty of transcripton (> 50X)            polyA site

                   MINIMUM CMV prom.                    your favorite gene

                                                                                                    Class 15
                                                                               25

      In vitro
      site-specific
      mutagenesis
                                                  (of coli DNA polymerase)




M13 = single stranded bacteriophage            M13 strand




                                      50% WT           50% mutant




                                                                             Class 15
                                                                                           26
                                         Get rid of WT genome:



                                                                 Enzyme that removes U from
Dut = deoxyuracil phophotransferase.                             U-containing DNA
Dut- cells accumulate dUTP,  DNA

Ung = uracil N=glycosylase.
Ung- mutants do not excise uracil from                           Mutant E. coli allows
DNA                                                              U to persist in DNA

Growth of plasmid template plasmid
on dut- ung- E. coli double mutant
cells  U in DNA

Synthesize mutant DNA using T.
Transfect heteroduplex into dut+ung+
E. coli.
DpnI sites
U gets removed from WT template
strand, leaving apyrimidinic sites,
these cannot replicate.

Mutant synthetic strand survives
normally.
                                                                          Apyrimidinic DNA
                                                                          formed and
                                                                          will not replicate



                                                                                         Class 15
                                                                                                       27
Use of DpnI to get rid of WT template.

                                               m

                                       m               m
                                   m
                                                       m
                                                                Template WT methylated DpnI sites
                                   m
                                           m       m
                                               m


    DpnI
                                               Synthesis


                                               m

                                       m               m
                                                                Newly synthesized mutant strand:
                                   m
                                                           m    its DpnI sites are not methyated,.
                                       m
                                           m       m
                                               m


                                                   DpnI treatment selectively destroys the
                                                   hemimethylated WT strand




                                                               Mutant strand greatly enriched.




                                                                                                     Class 15
                                              PCR-ligation-PCR mutagenesis                                    28
                                              Site to mutate


                                                    P
                                                   xP




                                                 PCR                             PCR


                                                  x
                                                  x



                                                        Blunt end ligate


                                                        x
                                                        x



                                                                     PCR again


                                                        x
                                                        x


                                                                    Cut with the 2 res. enz.
                                                                                               Clone into
                                                            x                                  framework
                                                            x
                                                                                               in vector

                              Note you can easily create an internal deletion this way.
Ali SA, Steinkasserer A., Biotechniques. 1995 May;18(5):746-50.
                                                                                                            Class 15
                                       RS1                RS2
                                                                                  29




Mutagenesis by overlap extension PCR

      PCR  fragment
        subsequent cloning
      in a plasmid




                                       RS1                      RS2




                                               Cut with RE 1 and 2


                                             Ligate into similarly cut vector

                                                                                Class 15
                                                                                                 30
             Random mutagenesis but in a limited region:
             Cassette mutagenesis by error-prone PCR

-----------------------------------------------------------    Original sequence
-----------------------------------------------------------    coding for, e.g., an enhancer

                                PCR fragment with high Taq
                                polymerase and Mn++ instead of Mg+  errors
 ------*--------*--*-**---------------*-----------*--*------
 -*------------------------*-*-*------------*------------*--




            Cut in primer sites and clone upstream of a reporter.



                    Pick colonies
                    Analyze phenotypes
                    Sequence




                                                                                               Class 15
                                                                                           31
             Cassette mutagenesis by “doped” synthesis
             Target = e.g., an enhancer element

-----------------------------------------------------------
                                                            Original enhancer sequence
-----------------------------------------------------------


-*------------------------*-*-*------------*------------*--
------*--------*--*-**---------------*-----------*--*------   Buy 2 doped oligos; anneal




           Clone upstream of a reporter.


                                                                 Doping = e.g.,
                   Pick colonies                                 85% G,
                   Analyze phenotypes                             5% A,
                   Sequence                                       5% C,
                                                                  5% T
                                                                 at each position


                                                                                         Class 15
                                                                                         32

             Measuring protein-protein interactions in vitro
             X=one protein Y= another protein

  Binding between (almost) purified proteins:-

  His6-X + HA-Y ; Bind to nickel ion column, elute, Western with HA Ab

  GST-X + HA-Y ; Bind to glutathione ion column, elute, Western with HA Ab

  His6-X +   35S-Y   (made in vitro) ; Bind Ni column, elute, gel + autoradiography.




(HA = flu hemagglutinin)


   BIAcore (or several other measurements)




                                                                                       Class 15
         Surface plasmon resonance (SPR)                                           33
         The binding events are monitored in real-time and it is not necessary
         to label the interacting biomolecules.




                     glass plate




http://home.hccnet.nl/ja.marquart/BasicSPR/BasicSpr01.htm




                                                                                 Class 15
                                                                        34




Surface plasmon resonance:

Detects binding of protein or
nucleic acids to molecules
immobilized on the opposite
surface.
                                 Flow cell carrying ligands or wash
Suitable for protein – protein
interaction, measure kinetics,
binding constants.
Use small amount (ug) and even
ligands in crude extracts

Popular instrument = Biacore




                                                                      Class 15
                                                  35




Protein-protein interactions in cells:


Co-immunoprecipitation (co-IP) from extracts


2-hybrid formation


FRET (Fluorescence resonance energy transfer)


Complementation readout




                                                Class 15
                                                                                                                  36
Co-immunoprecipitation

Most times not true precipitation, which requires about equivalent concentrations.
of antigen and antibody

Use protein A immobilized on beads (e.g., agarose beads)

Protein A from Staphylococcus aureus: binds tightly to Immunoglobulin G (IgG) from many species.

Does X interact with Y in the cell?

                  B                                                             Y           B
                              X                    incubate
          Y                                                                     X
                          Y       C
                                                                                        +
                  A                                                                     C
  X                                                                         X
              Cell extract                                                  Y               A
                                      A
                                              A    X                                                Y
                      Y       A                                         + Protein A                         B
                                              A    Y                                                X
                      X
          A                       A                                         A                           +
                                          A
                                                                                    A                   C
                      A                                             A
  A
                                                                                    A
                  A               Wash by centrifugation                                        X           A
                                                                        A
      A                           Elute with SDS                                A
              A                                                                                 Y
                                  Detect X, Y by Western blotting

                                                                                                                Class 15

								
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