Hey guys I'm attempting to express quite a large protein in XL1-Blue cells. The protein's approximately 140KD . It is a bacterial protein but not one found in E.Coli. I have seen evidence (SDS) that the protein has expressed quite well but I thought I'd ask more experienced people here have they any experience of expression and purifying large proteins from E.Coli?
Hi Harry,
it's good that you are seeing expression in XL1-Blue.
The one pice of advice I'd give you is to try strains that are meant for expressing proteins (XL1-Blue is a cloning strain, good for DNA, poor for protein). BL21 is one good expression strain that is commonly used. You don't say what vector you are using but as you are getting expression in XL1-Blue then it can't be one with a T7 promoter which would require BL21(DE3).
-Richard
I don't think there are any significant differences wrt optimal growth conditions between small and large proteins expressed in E. coli, at least not for laboratory-level work. Proceed as you would for smaller proteins. Expression levels will depend mainly on promoter and RBS strength, and biomass yield will depend more on the specific strain being used than on the finer points of medium composition, etc.
I agree with Richard. While you can sometimes get expression from cloning strains, they are not meant to be used for expression. I would do a western blot to make sure that what you are seeing is indeed your protein, but you should probably shift to an expression system such as BL21(DE3)
I agree, use BL21. I usually test at least 2-3 temperatures during the indcution like 37, or 30 deg for a couple of hours or induction at 20 deg over night.
In addition to all the suggestions, try auto-induction media!
If the protein is large and has a poor folding profile, co-expression of a chaperone protein (e.g. pGROESL) may help. Depending on protein stability, shorter induction at higher OD may be beneficial - growth trials are typically done at a variety of conditions to test this first before a large scale prep. Is it codon optimized for E.coli? Is it toxic? DIfferent commercial strains exist for various particular 'concerning' applications.
Tips for large proteins expression:
- use small tags only (e.g. His-tag, Strep-tag), avoid protein tags (GST, MBL, thioredoxin);
- use Rosetta expression strain and low IPTG concentration for induction of protein.
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