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Heterologous expression and purification of proteins in E. coli Rory Koenen Institut für molekulare Herz-Kreislaufforschung University Hospital of the RWTH Aachen [email protected] Tel. 35984 contents Things to consider when planning to express proteins in E. coli An introduction to protein purification This presentation can be downloaded soon from: http://www.imcar.rwth-aachen.de/ Click „Verschiedenes“ and then „Lehrmaterial“ Protein Expression prokaryotic gene expression • expression vector features • bacterial host features • solubility • affinity tags • general considerations Features of expression vector Origin: Essential for plasmid propagation. High copy vs low copy. Examples are pBR322, ColE1, pACYC T7 promotor/operator: Transcription initiation site for T7 RNA polymerase. Operator is binding site for Lac repressor in the absence of lactose. Ribosome binding site: needed for initiation of translation Multiple cloning site: facilitates cloning of the desired cDNA Resistance marker: essential for plasmid propagation Lac Repressor: needed for control of transcription and expression of cDNA not essential if the bacterial host already has a LacI gene Features of expression vector Origin T7 promotor/operator Ribosome binding site MCS Lac Repressor Expression vector 2961 bp Resistance Marker Novagen pET26b Features of bacterial hosts T7 RNA polymerase gene: Viral RNA polymerase that has high rates of transcription and is not needed for endogenous transcription of bacterial genes. Bacterial hosts that have the (DE3) lysogen are competent for T7-based vectors Some sophisticated strains have the T7 gene under LacI control Protease-deficient: some proteases degenerate the expressed protease. The BL21 strain is deficient for OmpT and Lon proteases. E. coli B or K strain-derived: can sometimes make a difference... Gene mutations for „oxidative cytoplasm“: may influence disulfide bond formation in some proteins Presence of the Lac Repressor gene LacI: needed for control of transcription and expression of cDNA Expression • cells are grown typically until mid-log phase • induction occurs by the lactose analog isopropyl-thiogalactoside: IPTG: non-metabolized lactose analog • Cells are harvested and prepared for purification Expression • protein can occur as soluble native protein (uncommon) • or as insoluble aggregates (common) • these insoluble aggregates are generally „stored“ in E. coli as so called inclusion bodies • inclusion bodies are dense and can be purified easily by centrifugation • they are very immunogenic and can be readily injected in animals • BUT they need to be dissolved, and the protein refolded to the native state • refolding can be very problematic solubilization and refolding • inclusion bodies can be dissolved using: – detergents (e.g. laurylsarcosine) – chaotropic salts (ureum, guanidine-HCl, arginine) – organic solvents or high/low pH buffers • refolding can take place by: – strong dilution in „native“ buffer – dialysis in „native“ buffer – binding to a column and perfusion with „native“ buffer Disulfides can be formed by additives like: – – – – – cysteine – cystine pair reduced and oxidized glutathione copper and o-phenanthroline hydrogen peroxide just air affinity/solubility tags • greatly simplify purification; especially for beginners/ non-experts • the alltime classic is the 6*histidine tag, which can be purified using metal chelation chromatography • another classic is the glutathione-S-transferase tag which binds strongly to GST-sepharose • other tags are chitin binding protein, maltose binding protein, polyarginine and many more. • solubility tags increase solubility of protein in cytoplasm: – examples are: thioredoxin, Nus. protein His His His His His His + Affinity chromatography resin resin affinity tags • the His-tag is a good choice because: - is often functionally neutral: no need for removal - metal chelation chromatography is cheap, easy and permits denaturing conditions - may support on-column refolding BUT - choice of column buffers is limited - protein mostly not so pure after single affinity chromatography step • the GST-tag is also good because: - it facilitates the fusion of small peptides - sometimes supports solubility of protein - a single purification step generally leads to very pure protein BUT - slow kinetics of binding - columns are expensive - cleavage often required but even more often quite problematic - GST very immunogenic and stable general considerations • N-terminus or C-terminal tags – what is known about protein function? – modified N-terminus can have big consequences: Met-RANTES – use a cleavable tag with a good protease like enterokinase or TEV • Codon usage – eukaryotic genes have different codon occurance as prokaryotic genes: Pro CCC and Arg AGA are common in humans but rare in E. coli • Protein toxicity / plasmid stability – toxic proteins do not express well and the E.coli cell will try to shut the expression down, sometimes by destroying the plasmid – even worse, during the growth, cells that express even traces of toxic protein will die, leaving you with cells that do not express anything – „leaky expression“ can be decreased using pLysS or pLys E plasmids in the host Protein Purification protein purification • Chromatography: – affinity: Ni-NTA, protein G/A (antibodies), GST-sepharose – kation exchange: positive charge by e.g. SP sepharose, Mono S – anion exchange: negative charge by e.g. Q sepharose, Mono Q – gel filtration: separation by size, Superose, Superdex, Sephacryl – hydrophobic interaction and reverse phase HPLC: hydrophobicity protein purification • ion exchange chromatography – separation by charge – protein charge depends on buffer pH – elution from column by pH shifts or shifts in ion strength (preferred) – permits a lot of different conditions – choice number one for routine purifications! Ion-exchange chromatography protein purification • gel filtration chromatography – separation by size – biggest proteins come first, smallest come last – permits a lot of different buffer conditions BUT – GF columns are expensive and fragile – sample size should be very small (<1% of column volume) – not a very good „first step“, more suitable for final refinement Gel filtration chromatography protein purification • reverse phase chromatography – separation by hydrophobicity – elution using organic solvents – high resolution and very quick – removes endotoxin contaminations – eluted proteins can immediately be analyzed using mass spectroscopy BUT – some proteins do not survive the buffer conditions – best performed on a HPLC system, which is an investment Purification of human PF4 from E.coli Slides: Alisina Sarabi expression of recombinant PF4 • plating of E. coli containing vector – Rosetta2(DE3) and pET26b/PF4 • growth in specialized medium • induction by IPTG and expression overnight • Centrifugation of the cells • Storage of pellet at -30°C Purity Purification strategy 3.Polishing High level purity 2.Intermediate purification Remove bulk impurities 1.Capture Isolate, concentrate, stabilize Preparation, Extraction, clarification Step SP Sepharose Capture Buffer A: 50mM NaAc pH 5,5 Buffer B: A + 2M NaCl pH 5,5 SP Sepharose Flow through After dialysis against 50mM NaAc pH5,5 the crude periplasmic solution containing the protein was purified by FPLC (0-2M NaCl gradient) using a HiLoad 16/10 SPSepharose™ Intermediate MonoS Buffer A: 50mM NaAc pH 5,5 Buffer B: A + 2M NaCl pH 5,5 CaptoS Buffer A: 50mM NaAc pH 5 Buffer B: A + 2M NaCl pH 5 Pool A13-B13 After dialysis against 50mM NaAc (pH 5,5 or 5) the protein was purified by FPLC (0-2M NaCl gradient) using a using a strong cation exchanger (MonoS™ 5/50 GL or HiTrap CaptoS) Polishing Reverse phase chromatograpy After dialysis 2 x against 1% acetic acid and 1 x against 0,1% TFA protein was purified by using a Recource™ RPC column Buffer A: 0,1% TFA Buffer B: 0,1% TFA + 90% ACN Coomassie staining Pool B8+B7 Polishing HPLC: Hypersil Gold (C-18) Silverstaining SDS Page Western Blot Mass spec (esi) PF4alt experiment. mass: 7805.8 D theoret. mass: 7806.1 D Thank you for your attention