Recombinant protein in bacteria by hcj

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									Manipulation of gene Expression
           in Bacteria

              EXPRESSION

EXPRESSION



              EXPRESSION
              Biotechnology
   The primary objective of gene cloning or
    genetic engineering is to express the cloned
    gene in a selected host organism
           Host System
 Bacteria
 Yeast
 Insect
 Mammalian cells
        GENETICALLY
    ENGINEERED PRODUCTS
   Some products produced by genetic
    engineering and are currently on the market

    –   Bt potatoes
    –   PRV Papaya
    –   Herbicide resistant corn
    –   Human growth hormone
    –   Insulin
    –   human interferon protein
    –   monoclonal antibodies
Gene expression in different
systems
 Bacterial genes being expressed in a
  eukaryotic system
 Eukaryotic genes are being expressed
  in bacteria
Expression vectors
 Vectors designed to overproduce
  specific gene products
 Use well-characterised RNA and protein
  synthesis systems

    – Lambda ZAPII
    – pTrc99A
    – pBluescript II SK+
    Manipulations to modulate
        gene expression
 The transcriptional promoter and terminator
  sequences
 The ribosome-binding site and the efficiency of
  translation in the host organism
 The number of copies of the cloned gene and
  whether the gene is plasmid borne or integrated into
  the genome of the host
 The intrinsic stability of the cloned gene protein within
  the cell
 The final cellular location of the synthesized foreign
  protein
Promoters
 Effective gene expression system
  requires a strong and regulatable
  promoter sequence upstream from the
  cloned gene
 Strong promoter has a high affinity for
  RNA pol
Regulated promoter
   Some cloned DNA may produce products that are
    toxic to the bacterial cell, and if the promoter driving
    production of such gene products were not regulated,
    the bacterial cell might be poisoned.
   High levels of continual expression of a cloned gene
    is often detrimental to the host cell because it creates
    an energy drain, thereby impairing essential host cell
    functions.
   Plasmids carrying a constitutively expressed gene
    may be lost after several generation since cells
    without plasmids can take over the culture
What is the best promoter type for You?

 Constitutive promoters always
  express your gene of interest and
  eliminate the extra complexity of adding
  an inducer.
 If you have a non-toxic gene and are
  not worried about the timing of your
  expression, using a constitutive
  promoter is easier.
Examples of Promoters
 T3
 T5
 T7
 Lac
 Trp
 35S from CMV
 Chicken  actin
Promoter…
 The 35S promoter for CMV is used to
  express viral genes in plants because a
  bacterial promoter or bacteriophage
  promoter cannot turn on genes in plants
 Mammalian expression uses the strong
  constitutive CAG promoter that consists of
  the chicken  actin promoter
        Tri Systems www.Qiagen.com
   A single vector is possible in three different
    expression systems
    – the T5 promoter/lac operator transcription-
      translation system for expression in E. coli
    – the p10 promoter for baculovirus-based
      expression in insect cells
    – the CAG (actin) promoter for expression in
      mammalian cells
Regulated expression vector
pTrc99A (Ref: Amann et al. Gene 69: 301-315 (1988))
   Designed for the regulated expression of genes in
    E. coli of fused and non-fused proteins
   Based on pKK233-2
   Origin of replication from pBR322
   Has a strong hybrid promoter trp/lac
   The lacZ ribosome binding site (RBS)
   MCS of pUC18
   rrnB transcription terminator
Ptac or Ptrc promoter
   Regulated hybrid promoter containing the –35
    promoter from the trp promoter and the –10
    region from the lacuv5 promoter
    – Lacuv5 is a variant of the lac promoter that contains
      altered nt seq in the –10 region is stronger than
      the wild type lac promoter and is repressed by the
      lac repressor and derepressed by IPTG or lactose
    – Does not require activation with CAP-cAMP
Lac
 In the absence of lactose, the E. coli lac
  promoter is repressed, turned off by the lac
  repressor protein
 Induction can be achieved by the addition of
  lactose or IPTG, prevents the binding of the
  repressor to the lac operator
 Catabolite activator protein (CAP) increases
  affinity of the promoter for RNA pol
Trp
 Trp promoter is negatively regulated, turned
  off by a tryptophan trp-repressor protein
  complex that binds to the trp operator and
  prevents transcription of the trp operon
 De repression, or turning on achieved by
  removing tryptophan or adding 3-indole
  acrylic acid
Consensus sequences
    – 5’-TATAAT-3’ for the –10 region
    – 5’-TTGACA-3’ for the –35 region


 The lacUV5 promoter has a consensus
  sequence at its –10 but not its –35 region
 The trp promoter has a consensus sequence
  at its –35 region but not its –10 region

    – By fusing the –10 region of the lac promoter and
      the –35 region of the trp promoter, called it the
      Ptac or Ptrc
pTrc99A
 lacI gene including its corresponding
  promoter (PlacI).
 Also carries the lacIq mutation for which
  helps to maintain repression of Plac
  transcription.
 Produces much higher levels of the lac
  repressor, thereby decreasing the leakiness
  under non-induced conditions, i.e.
  transcription of a cloned gene in the absence
  of an inducer
pTrc99A
   To produce the protein in pTrc99A, cells
    carrying the plasmid are grown to a
    moderate density and IPTG is added
Increasing protein production
   Strategy used to increase protein production
    in recombinant E. coli involves designing an
    expression vector with a Ts origin of
    replication and a regulated promoter
    – Replacing the origin of replication of the plasmid
      pPLc2833 with that from the plasmid pKN402
    – To make the plasmid pCP3
    – pCP3 contains the pL promoter, the -lactamase
      gene for amp resistance from pLc2833 and the
      origin of replication from pKN402
   cI repressor is present in the chromosome of the E.
    coli host
   The pL promoter is controlled by the cI repressor
    protein of the bacteriophage lambda, this switches
    off the transcription from pL promoter
   Cells that carry the pCP3 plasmid are grown first
    at 28C and then shifted to 42C
   At 28C the cI repressor is functional and turns off
    the pL promoter plasmid copy number is normal
   At 42C, the plasmid copy number increases and
    the ts cI repressor is inactivated
    Large scale production systems

 Temperature shifts take time and energy
 Chemical inducer e.g. IPTG is expensive
    Large scale production systems

   Two Plasmid system:

   In one plasmid:
    – The cI repressor was placed under the trp
      promoter (Ptrp) and inserted into a low copy
      plasmid
   In a second plasmid:
    – Have the cloned genes, e.g. -galactosidase and
      citrate synthase genes under the pL promoter
        Termination signals
 Two strong transcription terminators – T7
  from E. coli bacteriophage T7 and rabbit
  globin terminator sequence
 Contains signals for the polyadenylation of
  the mRNA transcript – to prevent read-
  through transcription to ensure stability of
  the expressed construct
Regulatory sequences
Ribosome binding site (RBS)
   The molecular basis for differential
    translation is the presence of a
    translational initiation signal called a
    ribosome binding site (rbs) in the
    transcribed mRNA
RBS
 Bacterial cells and human cells have
  different mechanisms for selecting
  translational start points on the mRMA
 This is an important consideration for
  producing human proteins in bacteria
            RBS- Bacteria
 Directed by a ribosome attachment sequence
  closely linked to the AUG initiator codon for
  translation.
 This ribosome attachment sequence consist
  of 6 – 8 nucleotides UAAGGAGG (Shine
  Dalgarno sequence) upstream of AUG
 This sequence is complementary to the 3’
  end of the 16S ribosomal RNA, AUUCCUCC
RBS
 Activity of a RBS can be influenced by the length
  and nucleotide composition of the spacer
  separating the RBS and the initiator AUG
 Bacterial mRNAs that do not have a close match
  to the consensus ribosome attachment sequence
  are not translated efficiently
 Generally, the stronger the binding of the mRNA
  to the ribosomal RNA, the greater the efficiency
  of translational initiation
RBS - Eukaryotes
 The ribosome binds to an mRNA
  through the 5’ terminal cap structure
 The ribosome then scans the RNA in
  the 5’ to 3’ direction until it located the
  first AUG in the mRNA
 If this AUG is surrounded by a suitable
  sequence, translation will begin
RBS - Eukaryotes
   This sequence in eukaryotes, called the
    Kozak sequence 5’-A/GCCACCAUGG
    which lies within a short 5' untranslated
    region, directs translation of mRNA
Conditions required for maximum
translation efficiency
 The RBS must be located at a precise distance
  from the translation start codon of the cloned gene
 Overproducing a human protein in E. coli requires
  customizing the gene to have a bacterial RBS
  appropriately spaced from the AUG codon for
  translation initiation
 The DNA sequence that includes the RBS through
  the first few codons of the gene must not contain
  nucleotide sequences that after transcription can
  fold back to form intra-strand loops, thereby
  blocking the interaction of the mRNA with the
  ribosome.
    Expression vector pKK233-2
 An ampicillin resistance gene
 The tac promoter
 The lacZ ribosome binding site
 An ATG start codon located 8
  nucleotides downstream from the RBS
 The transcription terminators T1 and T2
  from lambda
    Copies of the cloned gene
   Tandem gene arrays
    – level of gene expression is proportional to
     the number of copies of the transcribed
     gene in the host cell
STRATEGY
 Increase plasmid copy in the cell
 Clone multiple copies of the gene in tandem
  into a low copy number plasmid
Vector Preparation
   Unique site in vector is digested with AvaI
    (C_TCGGG)
   Filling in with DNA polymerase I
   EcoRI linker (GAATT_C) inserted
   EcoRI linker flanked by two AvaI sites
    (CTCGGAATTCTCGGG) is inserted into the
    plasmid
   The gene plus the signals are cloned into the
    EcoRI site by digesting with AvaI to give non-
    sticky ends so the genes are orientated in
    one direction
DNA integration into the host
chromosome
   When a gene is part of the host
    chromosomal DNA, it is relatively stable
    and consequently can be maintained for
    many generations
Factors to consider when
integrating a gene
 the chromosome integration site must not be
  within an essential coding gene
 the input gene must be under the control of a
  regulated promoter
 for integration the DNA sequence must have
  some sequence homology for recombination
  between the two DNA molecules
 chromosomal site about 50 nt
     Integration into the host
          chromosome
 The cloned gene inserted in the middle of a
  cloned segment of DNA (ab) from the host
  chromosome on the plasmid
 Homologous DNA pairing occurs between
  plasmid-bourne DNA regions a and b and the
  host chromosome DNA regions a’ and b’
 A double cross over event (X-X) results in the
  integration of the cloned gene
Stability of cloned gene
protein
 Small proteins and peptides are difficult to
  be stably expressed in E. coli
 These proteins can be stabilised by
  expressing them fused to a large protein
  such as mouse (DHFR) dihydrofolate
  reductase
Fusion Proteins

 Fusion proteins are constructed at the DNA
  level by ligating together the coding regions
  of two genes
 One gene is the host gene that produces a
  stable host protein and the other gene
  represents the cloned gene that will produce
  a foreign protein
 When the two genes are transcribed, the
  foreign protein produced will be covalently
  linked to the stable host protein
      Uses of fusion protein
 determine its location in a cell
 stability of the protein
 used as antigens and to generate
  antibodies
 Used to purify recombinant proteins
  through the technique of immuno-affinity
  chromatography
Histidine tag
 Fusion proteins can be designed with a His
  tag
 Can be placed at either N- or C- terminus
 Important that DNA is inserted in correct
  reading frame
His tag
 His tag at the N-terminus most commonly used,
  easiest to prepare
 When at the N-terminus only the 5’end of the
  ORF must be ligated in frame
 When at the C-terminus the insert must be cloned
  in frame with the ATG start codon and the 3’ His
  coding system
 At the C-terminus His tag facilitate cloning of full
  length

    Cleavage of fusion proteins
   Host protein

    – may affect the biological functioning of the
      target protein
    – may cause allergic responses
            Marker Peptide
   Saccharomyces cerivisiae plasmid
    construct for the production of
    interleukin-2 is joined to DNA encoding
    a marker peptide sequence
    (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)
    Function of Marker Peptide
 reduces degradation of the expressed
  interlukin-2 gene product and
 enables the product to be purified
    Purify recombinant proteins
   Monoclonal antibodies made against the
    marker peptide
   Immobilized on a polypropylene support
     and binds the fusion protein’s marker protein
   The secreted proteins are passed through the
    column containing the bound antibody
   The immunopurified fusion protein is then
    selectively eluted from the column
    marker protein is removed with bovine
    intestinal enterokinase.
   Some Fusion Protein System

Fusion Partner       Size   Ligand   Elution Condition
Protein A   14kb            IgG              Low pH
His tail    6-10aa          Ni2+             Imidazole
Strep-tag   10 aa     Streptavidin         Iminobiotin
          Metabolic load
 Increasing plasmid copy number or
  plasmid size
 Limited amounts of dissolved oxygen in
  the growth medium
 Overproduction of foreign proteins
 Jamming of export site
 Production of toxic products
     Overcoming O2 Limitation
   Use of protease-deficient strains
    – develop host strains that are deficient in the
      production of proteolytic enzymes
    – mutations in both the genes for the RNA pol
      sigma factor responsible for heat shock proteins
      synthesis (rpoH) and the gene for protease
      required for growth at high temperatures (degP)
    – secreted proteins had a 36-fold greater specific
      activity
    Overcoming O2 Limitation
Bacterial hemoglobin
 Vitreoscilla bacterium, GNB obligate aerobe,
  normally live in oxygen poor environments
  such as stagnant water.
 synthesizes a hemoglobin-like molecule
 gene for Hb-like protein cloned and expressed in
  E. coli, the transformant displayed higher levels of
  protein synthesis
    Overproduction of foreign
           proteins
 deplete the pools of certain aminoacyl-
  tRNA or even certain amino acids.
 increase translational errors 10-fold
 drain the host cell of it energy
 diminish the usefulness of the protein
 may cause the protein to be
  immunogenic
       Secretion of proteins
 Secretion of proteins in E. coli is mediated
  by an N-terminal signal sequence
 Promote proper folding and disulfide bond
  formation
 Direct toxic proteins out of the cell
     Jamming of export site
 Over expression of protein and export from
  cytoplasm to cell membrane or periplasm
  may jam export sites thereby preventing
  localization of essential proteins
 Over expressed protein may also interfere
  with proper functioning of the host cell
 Use of His-tag at the C-terminus to prevent
  interferences during N-terminal processing
Reference
 Glick and Pasternak. Molecular
  Biotechnology: Principles and Applications
  of Recombinant DNA 2nd Edition
 Chapter 6.

								
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