Hi there,

I guess the GST is on the Nter side of the fusion so free GST might be the result of premature stop codon and/or cleavage of the fusion protein (or degradation on Cter side) during expression. Do you observe free GST as well as free protein on gel (if respective MW are appropriate). To purify the fusion and get rid of GST, I suggest SEC or directly run affinity chromatography on GSH-agarose with on column protease treatment to specifically elute the protein of interest and keep GST on the solid phase.

I would look at your protein of interest sequence - is it optimized to be expressed in your particular vector? For example, if you are expressing human proteins in a bacterial system, you may have to optimize the codon usage or utilize different bacterial strains that can express proteins that are rare/absent in bacteria but present in human proteins. I remember using Rosetta strain bacteria:

Rosetta host strains are BL21 lacZY (Tuner™) derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains supply

tRNAs for the codons AUA, AGG, AGA, CUA,

CCC, GGA on a compatible chloramphenicol resistant

plasmid. The tRNA genes are driven by their native promoters.

http://wolfson.huji.ac.il/expression/bac-strains-prot-exp.html

Max: Many thanks for your great help! Your suggestions are extremely helpful! - Weimin

Hi there,

I guess the GST is on the Nter side of the fusion so free GST might be the result of premature stop codon and/or cleavage of the fusion protein (or degradation on Cter side) during expression. Do you observe free GST as well as free protein on gel (if respective MW are appropriate). To purify the fusion and get rid of GST, I suggest SEC or directly run affinity chromatography on GSH-agarose with on column protease treatment to specifically elute the protein of interest and keep GST on the solid phase.

Sounds like your protein is being cleaved off by an E. coli protease, you may want to try a E. coli cell line deficient in proteases (look at NEB lines), a different expression system (insect cells-tricky but very good), or add protease inhibitors during expression in E. coli (can get expensive).

I'll side with Gary.  Cellular protease is where I'd look first.  To test it, work fast, work cold, add inhibitors, and compare that directly vs working slowly and in the warm and without inhibitors.  Then you know. 

I agree wtih Max. You can tell me the details, then I may tell you the solution.

For sure add a good cocktail of protease inhibitors but also, as is the case with many fusion proteins in E.coli but GST especially in my experience, you will get  really good expression of your fusion partner but your  protein will be truncated. This is probably due to the poor fidelity of E.coli ribosomes-if they hit a 'snag' like a stretch of low usage codons or a stretch of amino acids that don't immediately begin to fold correctly they just 'give up' and the result is truncated proteins.

You can of course make life easier for the ribosomes by loading the cells with a better balance of tRNAs, as Max suggested, with Rosetta/RP/RIL etc. cells. In addition you can also improve the fidelity of the ribosomes by slowing the whole process down-reduce the temperature prior to induction and do a long slow induction 18-20°C overnight rather than 37°C for a few hours.

Richer media like TB instead of LB may also help as could auto-induction.

Good Luck

I had a similar problem last year and the Rosetta strain did not help. It seems some sequences just cannot be expressed in bacteria...

In my experience, the expresion of heterologous proteins fused with GST is usually so high that it is very frequent to obtain truncated forms of GST alone.

I found out from Life Technologies something like: "The structure of the GST tagged protein often degrades upon denaturation and reduction for protein Gel-electrophoresis (SDS-PAGE). As a result, electrophoresed samples often appear as a ladder of lower MW bands below the full-sized fusion protein."

But I have performed a gel-filtration of apparently degraded samples in SDS-PAGE and I have obtained several peaks corresponding the protein-GST and the GST alone. In my opinion, the reason is what Nick has explained, the process is very fast and the protein quantity is very high. I have had these results even in Rosetta strain therefore I prefer to reduce the temperature and the IPTG concentration for induction in order to improve the  quality of the protein synthesis

Hi Weimin,

Did you resolve this problem? we have the same problem not only for GST-fusion protein, but also for Nus-tag fusion protein. Actually, this is true for 3 different proteins that are our target.

Could you pls update?

Cheers

Hi Weimin,

I have exactly the same problem with SUMO-fusion protein. I got huge amount of free SUMO protein compared with my SUMO-fusion protein after IPTG induction. I had these results even in Rosetta strain, low temperature (20 degrees) and low IPTG concentration for induction during 18 hours.

Could you please provide me how can I confirme if my plasmid is not mixed with empty SUMO vector?

Viviane, you could try the following: re-streakout the cells and pick several single colonies and do mini preps to get plasmid DNA, and use restriction digestion to confirm they contain the insert before doing the expression. Make sure you are using good sterile technique and aerosol barrier tips for all steps since your pipettes could be contaminated with bacteria and or plasmid! If the cloned protein is at all toxic, the empty vector containing cells will rapidly out grow the recombinant cells.

Have you tried adding glucose to 1% w/v final concentration to all your plates and all your cultures? It sounds simple but really we have seen the difference between no usable protein and 10s of mgs/litre by simply helping to block lac promoter with glucose.

Don't use plates/transformations in B strains (and most derivatives) older than one week for unstable constructs.

Auto-induction media can also help with this problem. P.S. for auto-induction use glucose on plates and starter culture but NOT in final expression culture.

If you have truncations you could also do SEC or IEX to seperate the full length from the truncated as already suggested and/or add a second affinity tag at the c-terminus of your protein to (by definition) purify only full length. N.B. The second option may not work with some some fusions like GST as they are known to dimerize.

If you solve it please let us know what worked for you!