Heterologous expression and purification of proteins in E. coli

Transcription

1 Heterologous expression and purification of proteins in E. coli Rory Koenen Institut für molekulare Herz-Kreislaufforschung University Hospital of the RWTH Aachen Tel

2 contents Things to consider when planning to express proteins in E. coli An introduction to protein purification This presentation can be downloaded soon from: Click Verschiedenes and then Lehrmaterial

3 Protein Expression

4 prokaryotic gene expression expression vector features bacterial host features solubility affinity tags general considerations

5 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

6 Features of expression vector Origin T7 promotor/operator Ribosome binding site MCS Lac Repressor Expression vector 2961 bp Resistance Marker

7 Novagen pet26b

8 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

9 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

10 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

11 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

12 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 +

13 Affinity chromatography resin resin

14 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

15 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

16 Protein Purification

17 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

18 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!

19 Ion-exchange chromatography

20 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

21 Gel filtration chromatography

22 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

23 Purification of human PF4 from E.coli Slides: Alisina Sarabi

24 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

25 Purification strategy Purity 3.Polishing High level purity 2.Intermediate purification Remove bulk impurities Preparation, Extraction, clarification 1.Capture Isolate, concentrate, stabilize Step

26 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 SP- Sepharose

27 MonoS Intermediate CaptoS Buffer A: 50mM NaAc ph 5,5 Buffer B: A + 2M NaCl ph 5,5 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)

28 Polishing Reverse phase chromatograpy Buffer A: 0,1% TFA Buffer B: 0,1% TFA + 90% ACN After dialysis 2 x against 1% acetic acid and 1 x against 0,1% TFA protein was purified by using a Recource RPC column Coomassie staining Pool B8+B7

29 HPLC: Hypersil Gold (C-18) Polishing

30 Silverstaining SDS Page Western Blot

31 PF4alt experiment. mass: D theoret. mass: D Mass spec (esi)

32 Thank you for your attention