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Download Lecture 14 Cloning and Expression E. coli Expression System
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Lecture 14 Cloning and Expression Expression Systems Bacterial: Escherichia coli , Bacillus subtilis Mammalian : animal cells Others: Saccharomyces cerevisiae, Insect cells Factors considered when choosing an expression system • • • The size of protein The amount of protein needed Whether active protein is needed E. coli Expression System 1. Choosing a promoter and vector system 2. Fusion proteins 3. Insoluble proteins 4. Optimization of expression of foreign proteins 5. Competent cells and transformation The Nobel Prize in Physiology or Medicine 1965 "for their discoveries concerning genetic control of enzyme and virus synthesis" François Jacob Institut Pasteur Paris, France 1920b From the early 1950's Jacques Monod, at the Pasteur Institute in Paris, had been studying the ability of E. coli to adapt to grow on different sugar sources. In particular, he studied the ability of E. coli to metabolize lactose. This depended on the ability of the cells to synthesize the enzyme ß-galactosidase. Later on, it was found that ßgalactoside permease was needed, too). André Lwoff Institut Pasteur Paris, France 1902-1994 Jacques Monod Institut Pasteur Paris, France 1910-1976 Operon model and lac operon (i) Operon model of Francois Jacob and Jacques Monod: the model consists of a regulator gene, operator site and a set of structural genes in order to explain the parallel regulation of β-galactosidase, permease, and transacetylase. (ii) Lactose operon or lac operon: i encodes gene for repressor, o is operator site, z, y, a are structural genes for β-galactosidase, permease, and transacetylase, respectively. p is promoter site. z, y, a give a polycistronic (or polygenic) transcript. The lac operator, nearly perfect inverted repeat, 2-fold rotational symmetry. An operon is a cluster of genes that are transcribed as a single mRNA. Genes in an operon code for a diffusible gene product which may be a polypeptide or an RNA molecule. Lac repressor binding to operator DNA (Kd=10-13) or 0.1 pM. The binding of the α-helix into the major groove of DNA. E. coli genome has one lac operator (blue) but many pur operator (red). 20 pur or purine operators for 19 operons and 25 genes When glucose is scarce, E. coli use lactose as the carbon source. β-galactosidase will be induced to break down lactose. $ The enzyme activity can be measured by X-gal assay. In the presence of lactose, β-galactosidase constitutes 6.6% of total protein synthesized. (i) (ii) in the absence of lactose, repressor binds at o site and represses transcription from the lac operon. IPTG binds to the repressor so that it cannot bind to O site, and transcription can start. DNA Inducer such as IPTG (isopropylthiogalactoside) or allolactose will bind to the repressor and alter its binding domain so that it cannot bind to o site, and thus RNA pol can transcribe the lac mRNA. Domain for a large class of proteins including taste proteins. IPTG IPTG-inducible vector pGEM series (Promega), pGEX series (Pharmacia), pKK223-3 (Pharmacia), pMEX (US Biochemicals), pTrc99A (Pharmacia), pMAL (New England Biolabs). IPTG-inducible promoter: pGEM Used for in vitro transcription and protein expression. The vector encodes the LacZ α-peptide and β-lactamase and contains restriction site, lac O, T7, SP6 initiation sequence, promoter with efficient ribosome-binding site (ATG + Shine-Dalgarno sequence, complementary to 16S rRNA 3’-terminal), t terminator or RNase III site (stabilize mRNA, facilitate translation). IPTG-inducible promoter can lead to an expression level exceeds 30% of total protein. E. coli strains: The strain should carry either the lacIq or lacIq1 allele. If the plasmid contains lacIq1 then any strain is fine. lacIq or lacIq1 generate 10x repressor to prevent any leaky expression. Promega Bacteriophage T7 promoter Bacteriophase T7 RNA polymerase, unlike E. coli RNA polymerase, is not inhibited by rifampicin. Thus host genes can be turned off. The T7 polymerase can only recognize the T7 promoter. The T7 polymerase is a processive enzyme that will transcribe around a circular plasmid several time and may transcribe genes that are not efficiently transcribed by E. coli enzyme. The bacteriophage T7 expression system (1) T7 polymerase either on a λ vactor, a plasmid, or inserted into E. coli chromosome. (2) a plasmid vector equipped with a T7 promoter upstream of the gene to be expressed. (3) pET series from Novagen The pET expression system T7 phage expression vector: Expression vector containing the bacteriophage T7 promoter carrying colicin E1 replicon (colE1), MCS, with insert under control of f10 promoter for T7 RNA polymerase or under the control of T7lac promoter. E. coli strains: HMS174(DE3) or BL21(DE3) Vectors: pET series (Novagen: www.novagen.com). Expression system: (i) In lDE3 lysogens the T7 pol gene (T7 gene 1) under control of lacUV5 promoter, (ii) pET.pLysS or pLysE encode lysozyme which inhibits T7 pol under uninduced condition. Fusion proteins • • • • Attaching target protein to a domain to provide a tag Topogenic signals to direct the protein to a cellular site Adding “carrier” sequences to protect the protein Adding “carrier” sequences to improve solubility Affinity purification of fusion protein fused partner β-galactosidase ProteinA Glutathione-S-transferase Maltose-binding protein Chloroamphenicol Acetyltransferase Carbonic anhydrase Cellulose-binding protein Poly(histidine) FLAG Poly(arginine) Poly(cysteine) Poly(phenylanaline) affinity resin APTG IgG glutathione crosslinked amylose Chloroamphenicol- Eluent sodium borate (pH 10) 0.5 M acetic acid 5 mM GSH 10 mM maltose chloramphenicol Sepharose sulfonamide-affinity resin Tris-sulfate cellulose immobolized Ni anti-FLAG S-sepharose water imidazole EDTA NaCl gradient thiopropyl-sepharose mercaptoethanol or DTT phenyl-sepharose ethylene glycol Cleavage of fusion proteins Chemicals: cyanogen bromide and 70% formic acid (-M/) formic acid (70%) and heat (-D/P) hydroxylamine pH9 and heat (-N/G-) Enzymes: ala-64 subtilisin (-GAHR/) clostripain (-R/ and -KR/) collagenase (-PV/GP-) enterokinase (-DDDDK/) Factor Xa (-IE(D)GR/) renin (-PFHL/L-) α-thrombin (-LVPR/GS-) trypsin (-R/ or -K/) Tev protease (-ENLYFQ/G-) Glutathione-S-transferase (GST) gene fusion system * * * * A tac promoter for chemically inducible, high-level expression. An internal lac Iq gene for use in any E. coli host. Very mild elution conditions for release of fusion proteins from the affinity matrix. PreScission™, thrombin, or Factor Xa protease recognition sites for cleaving the desired protein from the fusion product. GSH is attached to Sepharose 4B by coupling to the oxirane group using epoxy-activation. The binding capacity is about 8 mg/ml resin. The elution condition is mild, 10 mM GSH. Tac promoter is a functional hybrid derived from the trp and lac promoter (see de Boer et al., PNAS 80, 21-25, 1983 tac_promoter TGACAATTAATCATCGGCTCGTATAATGT Cellulose-binding domain Beta sheet in CBD. W17 W54 and W72 in contact with cellulose. Cellulose-binding domain vectors pET with 3 CBD sequences (with His-tag, GST-tag, Ek=enterokinase, Xa=factor Xa, Thrombin cutting site). Cellulose-binding domain $ SDS-PAGE (4-20%), pET34 CBD BAP in BL21(DE3) (37˚C, 1 total, 2 total+, 3 soluble 1.5 h, 4 soluble 3h, 5 soluble 4.5h, 6 insoluble 1.5 h, 7 insoluble 3h, 8 insoluble 4.5 h); pET36 BAP BL21(DE3)pLysS (16h 30˚C, 1 total, 2 total+, 3 media, 4 media+, 5 periplasmic +, 6 soluble +, 7 insoluble. The FLAG technology N- and C-cloning. Kit: (pFLAG vector, control vector, anti-FLAG monoclonal antibody, biotinylated anti-FLAG antibody, FLAG affinity resin and column, control FLAG-BAP, sequencing primers, FLAG peptide, host strain, enterokinase). 3 hosts (E.coli, yeast, mammalian cells). The FLAG technology The FLAG technology www.sigma-aldrich.com The FLAG technology Transformation Chemical Methods: calcium chloride, other variations include reducing agents, other divalent cations, freezing and thawing, heat shock, organic solvent shock etc (Hengen, 1996) Physical Methods: electroporation Competent cells Competent cells Improvement of competent cells: adding tRNA genes that encode for rare E. coli codons