E. Coli Expression System (T7 Promoter)

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E. Coli Expression System(T7 Promoter)

• ? ? ?

2003. 4. 10 (?)
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• Metagenome
• (metabolic enzyme genome)
• Construct libraries from total community genomic
  DNA
• Isolate genes of interest by phenotypic analysis
  in a surrogate host

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Recombinant protein expression in E. coli

• E. coli
• Gram negative bacterium
• Fast growth,
• High cell density,
• Well characterized genetics
• High availability of vectors and mutated host
  strains
• Required factors
• Selection marker
• Promoter
• Ribosome binding site
• Termination signal
• Origin of replication
• Coding (c)DNA

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Introduction

• Comparison of expression systems

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• Lac operon

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• Bacteriophage T7
• 39,937 bp linear dsDNA genome
• 56 genes thought to 60 proteins
• Primary function assigned to 33 genes
• Over 50 regulatory elements
• 62nm diameter particle, short tail

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• Protein Folding GroE structure

The core structure of chaperonin consists of two
identical rings composed of seven GroEL subunits.
Unfolded proteins bind to the central cavity.
Bound ATP molecules can be identified by their
red oxygen atoms (spacefill). The quaternary
structure is shown from (a) the side, and (b) the
top. (c) During folding, the size of the central
cavity of one of the rings increases and the end
is capped by a protein containing seven GroES
subunits. Highlighted in green is one of the
GroEL subunits. The process by which this protein
is believed to function is shown in the next
slide.
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The unfolded polypeptide enters the central
cavity of chaperonin, where it folds. The
hydrolysis of several ATP molecules is required
for chaperonin function
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Gateway Technology

• High efficiency cloning (over 99)
• Generation of native proteins, or N- or
  C-terminal fusion proteins
• transfer by recombination into Entry Vectors of
  genes from genomic or cDNA libraries
• prepared in attB-containing GATEWAY vectors
• cloning of restriction enzyme-generated
  fragments including cDNA and genomic
• libraries and PCR fragments into Entry Vectors
  by standard recombinant DNA methods.

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• attL x attR excision reaction

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TOPO cloning

• DNA topoisomerase I from Vaccinia virus binds to
  double strand DNA at specific sites and
  cleaves the phosphodiester backbone after
  5'-CCCTT in one strand (Shuman, 1991).
• The energy from the broken phosphodiester
  backbone is conserved by formation of a covalent
  bond between the 3' phosphate of the cleaved
  strand and a tyrosyl residue (Tyr-274) of
  topoisomerase I.

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• Topoisomerase is better than ligase
• Ligase is often contaminated by nuclease
• Topoisomerase I protects the vector ends from
  exonuclease to maintain the integrity of the
  vector
• Much higher probability event than a typical
  ligation in three molecules Only two molecules
  must come into contact for ligation to occur-the
  TOPO-activated vector and the insert.

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T7 Expression system

• pET system
• The pET System is the most powerful system yet
  developed for the cloning and expression of
  recombinant proteins in E. coli.
• Target genes are cloned in pET plasmids under
  control of strong bacteriophage T7 transcription
  and (optionally) translation signals expression
  is induced by providing a source of T7 RNA
  polymerase in the host cell.
• T7 RNA polymerase is so selective and active
  that, when fully induced, almost all of the
  cells resources are converted to target gene
  expression the desired product can comprise more
  than 50 of the total cell protein a few hours
  after induction.

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• vector
• Transcription vectors are designed for expression
  of target genes that already carry their own
  prokaryotic ribosome binding site and AUG start
  codon. There are only three transcription
  vectors.
• Translation vectors contain the highly efficient
  ribosome binding site from the phage T7 major
  capsid protein and are used for the expression of
  target genes without their own ribosomal binding
  site.
• Solubility
• provide for fusion to a polypeptide that itself
  is highly soluble e.g. glutathione-
  S-transferase (GST), thioredoxin (Trx), N
  utilization substance A (NusA)
• Provide for fusion to an enzyme that catalyzes
  disulfide bond formation (e.g. thioredoxin, DsbA,
  DsbC),
• provide a signal sequence for translocation into
  the periplasmic space. When using vectors
  designed for cytoplasmic expression, folding can
  be improved in hosts that are permissive for the
  formation of disulfide bonds in the cytoplasm
  (e.g. trxB and gor mutations,

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• Cloning strategies
• All of the pET translation vectors contain
  translation stop codons
• Produce native proteins without fusions
• Produce native proteins without fusions after
  protease cleavage
• Gateway technology
• Tppo cloning
• Regulating Protein Expression
• The T7lac promoter to control basal expression
• pLysS and pLysE hosts
• Another way of providing additional stability to
  target genes
• In host strains, a compatible chloramphenicol-resi
  stant plasmid that provides a small amount of T7
  lysozyme, a natural inhibitor of T7 RNA
  polymerase
• Media containing glucose
• low basal expression levels by supplementing the
  medium with glucose.

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• Vector
• pET vector
• Gateway destination vector
• T7 promoter for expression
• lac O, lac I tighter regulation of
  transcription
• origin of replication pBR322 (minimal basal
  expression)
• selectable marker Ampr , Cmr
• ccdB control of cell death (Zero Background
  cloning)
• attR1, attR2 efficient recombination with any
  attL-
• flanked Gateway Entry
  vector
• RBS ribosome binding site
• Start codon (ATG)
• Tag, Fusion 6xHis, V5 Epitope, GST
• T7 terminator

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• TOPO destination vector
• Linearized, topoisomerase Iactivated pET
  expression vector
• 5 min directional cloning
• lac O, lac I tighter regulation of
  transcription
• origin of replication pBR322 (minimal basal
  expression)
• selectable marker Ampr
• T7 promoter for expression
• RBS
• Start codon (ATG)
• Tag, Fusion 6xHis, Xpress Epitope, EK
  (Enterokinase), etc
• T7 terminator

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• pRSET (A, B, C)
• origin of replication pUC ori
• selectable marker Ampr
• T7 promoter for expression
• RBS
• Start codon (ATG)
• Tag, Fusion 6xHis, Xpress Epitope, EK
  (Enterokinase), etc
• polylinker (MCS) BamHI, HindIII, EcoRI, etc
• T7 terminator

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• Hosts for expression

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• Media and Reagents
• LB Broth
• LBAmpicillin Agar
• NZY Broth
• SOB Medium
• SOC Medium
• Agarose Plates
• TE Buffer
• NZY Top Agar
• 2 SDS gel sample buffer
• SM Solution

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pRSET vector

• DescriptionThe pRSET vector is designed for
  high-level prokaryotic expression controlled by
  the strong bacteriophage T7 promoter. Expression
  is induced by the production of T7 RNA polymerase
  in the BL21(DE3)pLysS host E. coli cells. These
  cells also produce T7 lysozyme to reduce basal
  expression of target genes. The pRSETvector
  offers
• High-level expression from the bacteriophage T7
  promoter
• T7 gene 10 sequence to provide protein stability
  
• N-terminal polyhistidine (6xHis) tag for rapid
  purification with ProBond resin
• N-terminal Xpress epitope for protein detection
  with the Anti-Xpress Antibody
• Enterokinase cleavage site for removal of fusion
  tag
• f1 origin for ssDNA rescue to allow easy
  sequencing and mutagenesisA set of three vectors
  is provided (A, B, and C). Each has the
  N-terminal tag coding sequence in a different
  reading frame relative to the multiple cloning
  site to simplify in-frame cloning of your gene.

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pCRT7/CT TOPO TA Cloning Kit

• DescriptionThe pCRT7/NT and pCRT7/CT TOPO TA
  Cloning Kits offer 5-minute cloning of
  Taq-amplified PCR products directly into a
  prokaryotic expression vector. The vector in each
  kit, pCRT7/NT-TOPO or pCRT7/CT-TOPO, is
  provided linearized and activated with
  topoisomerase I to allow 5-minute TOPO Cloning
  with gt85 cloning efficiency. Both vectors carry
  the bacterial phage T7 promoter for high-level
  protein expression in E. coli. To simplify
  protein purification and detection, the vectors
  also include the following features
• pCRT7/NT-TOPO
• N-terminal Xpress epitope for detection with an
  Anti-Xpress Antibody
• N-terminal polyhistidine (6xHis) tag for
  purification using ProBond resin and detection
  with
• an Anti-HisG Antibody
• Enterokinase cleavage site for efficient removal
  of the N-terminal fusion tag

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pCRT7/NT TOPO TA Cloning Kit

• pCRT7/CT-TOPO
• C-terminal V5 epitope for detection with an
  Anti-V5 Antibody
• C-terminal polyhistidine (6xHis) tag for
  purification using ProBond Resin and
• detection with an Anti-His(C-term) Antibody