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Protein purification
- 2. What are your Goals? • How much protein will you need? • Natural source or recombinant? • How pure? • How stable is the protein? • Will you be making mutations? • Do you have an activity to protect?
- 3. Purification of Proteins • Preparation of proteins –natural source or recombinant • Soluble, membrane-bound or inclusion bodies • Recombinant - tags for expression and identification - mutagenesis - optimization of codon usage to increase expression • Capture from bulk proteins • Affinity • Ion exchange • Enrichment • Affinity • Ion exchange • Polishing Size Exclusion Chromatography Often 2-3 columns steps will be required to obtain purity Yields decrease with each step
- 4. Expression of Recombinant Proteins • Choice of E. coli strain depends on expression system • T7 expression requires T7 polymerase also be expressed (eg (DE3) lysogens) • lac or tac promoters require LacIQ gene be present on plasmid or in host • Host mutations eg Protease deficient strains, rare codon tRNAs • Choice of promoter system • T7 strong expression that can be modulated by pLysS or pLysE plasmids • Lethal genes can be tightly controlled with arabinose promoter (pBad) • tac promoter gives intermediate expression modulated with IPTG • Choice of tags for purification or to increase solubility • His tags and GST tags allow easy purification • MBP improves solubility and allows purification • Slowing protein expression with lower inducer concentrations and low temperature (22-18°C) incubation can improve solubility
- 5. Preparation of Protein Extracts • Use protease inhibitors and keep on ice to prevent proteolysis • Keep cell pellets frozen if not using immediately • Minimize time between lysis and purification. Once the cells are broken open you must continue to purify • Cells may be lysed by sonication, French press or Emulsiflex. • Centrifuge to remove unbroken cells and cell debris French Press Cell Lysis Sonicator Emulsiflex
- 6. Stage 1 Purification –The Capture GST-Fusion Purification Chromatograph SDS-PAGE GST fusion flowthrough GST fusion
- 7. Stage 2/3 – Enrichment/Polishing • Size Exclusion Chromatography • Separates proteins based on size • Assessment of oligomeric state of protein. Is there aggregation? • Assessment of purity • Buffer exchange aggregates dimer monomer