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.

An Introduction to Protein Purification

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?

What to consider before starting:

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

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)

General Considerations

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).

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

PL and temperature sensitive 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.

The lac System

•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 plasmidsA plasmid copy of lacIq is required for regulated expressionIPTG (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.

Place your gene here

LacI+IPTG

Plac, Ptac, Ptrc, PlacUV5

The PL System

“inducer” is cheap!

Difficult to get rapid temperature shifts on large scale

Moderate levels of over-expression

Not as leaky as lac system

cIts43oC

PL

denatured

Place your gene here

T7 Expression systems

•The best OVER-expression system available30-60% total cell protein not uncommon but often have inclusion bodiesThe 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

T7 RNA Polymerase

LacI+IPTG

Plac

PT7

Place your gene here

T7 RNAPT7 lysozyme