Re: [ccp4bb] Using chaperones to boost expression in E. coli
Hi, I wanted to thank everyone for their responses to my inquiry. It's good to see that there have been positive examples, and also that one should not be discouraged from trying by initial negative results. Thanks again, and have a nice weekend, Michael From: Olga Kolaj [mailto:olga.ko...@gmail.com] Sent: Tuesday, April 26, 2011 5:17 AM To: Kothe, Michael Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Using chaperones to boost expression in E. coli Michael, We have put together a review about use of folding modulators to improve expression in E. coli some time ago which might be useful to you (Kolaj et al Microb Cell Fact. 2009 Jan 27;8:9). Here is the link: http://www.ncbi.nlm.nih.gov/pubmed/19173718 http://www.ncbi.nlm.nih.gov/pubmed/19173718 Regards On Fri, Apr 22, 2011 at 4:28 PM, Kothe, Michael michael.ko...@genzyme.com wrote: Dear ccp4bb, I am curious to hear of examples where the expression of well-behaved protein was achieved by the coexpression of chaperones in E. coli. I know the appropriate strains and vectors exist, but I can't remember hearing of a successful case. I have heard anecdotally of several cases where it was tried without success (including one attempt I made myself). I also heard of concerns that the chaperones might copurify with the (now soluble) protein of interest and are difficult to remove. Thanks, Michael
Re: [ccp4bb] Using chaperones to boost expression in E. coli
Michael, We have put together a review about use of folding modulators to improve expression in E. coli some time ago which might be useful to you (Kolaj et al Microb Cell Fact. 2009 Jan 27;8:9). Here is the link: http://www.ncbi.nlm.nih.gov/pubmed/19173718 http://www.ncbi.nlm.nih.gov/pubmed/19173718Regards On Fri, Apr 22, 2011 at 4:28 PM, Kothe, Michael michael.ko...@genzyme.comwrote: Dear ccp4bb, I am curious to hear of examples where the expression of well-behaved protein was achieved by the coexpression of chaperones in E. coli. I know the appropriate strains and vectors exist, but I can’t remember hearing of a successful case. I have heard anecdotally of several cases where it was tried without success (including one attempt I made myself). I also heard of concerns that the chaperones might copurify with the (now soluble) protein of interest and are difficult to remove. Thanks, Michael
Re: [ccp4bb] Using chaperones to boost expression in E. coli
Hi Michael, It worked for me with one viral enzyme but did not work with 2 others. In the successful case, I used Takara's chaperone plasmids to screen for the best composition of chaperones. The GroEL-GroES improved the yield of the soluble enzyme, but I got same activity (per 1L of media) as before. The plasmid with chaperone tig gave the biggest increase in activity (~4-8 fold). Still, the yield was rather low, in a range of 1 mg of purified protein per 1L of media. I also had to optimize the expression time and temperature to get best results. They were ~14 h at 17-20C. Interestingly, the protein failed to express in insect cells (was too poisonous for them), so the combination of a solubilizing domain, chaperones and Ecoli expression were the only available option for me. Regards Alex On Apr 22, 2011, at 8:28 AM, Kothe, Michael wrote: Dear ccp4bb, I am curious to hear of examples where the expression of well-behaved protein was achieved by the coexpression of chaperones in E. coli. I know the appropriate strains and vectors exist, but I can’t remember hearing of a successful case. I have heard anecdotally of several cases where it was tried without success (including one attempt I made myself). I also heard of concerns that the chaperones might copurify with the (now soluble) protein of interest and are difficult to remove. Thanks, Michael
Re: [ccp4bb] Using chaperones to boost expression in E. coli
As with all these things, YMMV. In our experience, chaperone co-expression can help not at all, can produce more folded protein or can produce more soluble protein, with co-purifying chaperone. With GroEL/S at least, chaperone release is easy (add ATP) and sometimes results in the liberation of folded protein, sometimes not. One case where for us chaperones made an enabling difference: Acta Cryst. (2005). F61, 403–406 Like most things, worth trying! cheers Shaun
