General Considerations Expression systems Protein Fusion / Purification

1. An Introduction to Protein Purification General Considerations Expression systems Protein Fusion / Purification Overview of Standard Methods Examples of Purification Schemes General References: Current Protocols in Protein Science Wiley & Sons, NY (the big “blue” book) Guide To Protein Purification M.P. Deutscher, Ed. Methods in Enzymology Vol. 182 (also other Meth. Enz.) Also the protocol manuals for the “fusion systems” as quite detailed.

2. What to consider before starting: • 1) How much protein is required? • 2) How ‘pure’ does it have to be? • 3) Does the final product need to be ‘functional’? • 4) How often will the purification be carried out? • If the gene is cloned, is it necessary to move it into an “expression system”? • If the gene is not cloned, would it be advantageous to clone it before trying to purify the protein?

3. General Considerations What is the nature of the protein being purified? Monomeric, multimeric, complex of several polypeptides What is the subcellular location of the protein? Cytoplasmic, periplasmic, peripheral or integral membrane, excreted Does it have specific properties to consider? DNA binding protein, specific co-factors

4. General Considerations An assay to follow the protein through the purification? 1) an enzymatic or other functional assay (specific DNA binding) 2) Western blotting if you have antibodies 3) recognizable band on an SDS-PAGE gel usually possible if the protein is over-expressed (but not so good if you are trying to purify functional protein) Proteins are typically much more ‘fragile’ than nucleic acids (i.e. they can often be easily denatured). Do not vortex; when mixing or resuspending do it gently and avoid introducing air (i.e. bubbles/foam).

5. Expression Systems • A cloned gene in a tightly regulated expression system that can significantly overproduce the protein of interest can greatly facilitate the purification. • There are several common systems: • Lac promoters and lacI repressor • l PL and temperature sensitive l cI repressor • T7 promoters and T7 polymerase • tetR and tet-lac hybrid systems The are also “fusion systems” that allow for affinity purification of a protein. These typically use one of the above expression systems.

6. The lac System +IPTG LacI Place your gene here Plac, Ptac, Ptrc, PlacUV5 • These lac promoters are all leaky • (This can be a problem for even mildly toxic genes) • The fold induction is never great (30-50X max) • Chromosomally encoded lacIq is insufficient to regulate lac promoters on high copy plasmids • A plasmid copy of lacIq is required for regulated expression • IPTG (the preferred inducer) is expensive and this is a consideration for large scale preps. • If a plasmid copy of lacIq is being used (i.e. a lot of LacI around) then higher amounts of IPTG are required for full induction.

7. Thel PLSystem denatured 43oC lcIts Place your gene here l PL • “inducer” is cheap! • Difficult to get rapid temperature shifts on large scale • Moderate levels of over-expression • Not as leaky as lac system

8. T7 Expression systems +IPTG LacI T7 RNA Polymerase Plac T7 lysozyme T7 RNAP Place your gene here PT7 • The best OVER-expression system available • 30-60% total cell protein not uncommon but often have inclusion bodies • The leaky lac promoter can be overcome with T7 lysozyme to reduce toxicity problems • For very toxic genes, T7 RNAP can be provided by super-infection with T7 phage or l phage carrying the T7 rnap genes • Strains are very unstable • plasmid should be transformed into expression strain every time