Transcript No Slide Title

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.
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?
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
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).
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.
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.
The l PL System
denatured
lcIts
43oC
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
T7 Expression systems
+IPTG
LacI
T7 RNA Polymerase
Plac
T7 RNAP
T7 lysozyme
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