Protein purification workflow

1. 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