From owner-cellbiol@net.bio.net Tue Feb 18 22:00:00 1997 Path: biosci!rutgers.rutgers.edu!uwm.edu!newsfeeds.sol.net!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!cam-news-hub1.bbnplanet.com!news.bbnplanet.com!howland.erols.net!torn!resunix.sickkids.on.ca!news From: Randy Willis Newsgroups: bionet.cellbiol,bionet.microbiology Subject: Re: GST-fusion system hints and questions Date: Tue, 18 Feb 1997 18:30:24 -0500 Organization: The Hospital For Sick Children Lines: 49 Message-ID: <330A3B90.41C6@gandalf.psf.sickkids.on.ca> References: NNTP-Posting-Host: gollum.psf.sickkids.on.ca Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0 (X11; I; IRIX 5.3 IP12) To: "Bart K. Kwieciszewski" Xref: biosci bionet.cellbiol:6684 bionet.microbiology:8969 Bart, I have to admit to not having had a lot of experience specifically with the GST fusion systems, but I am willing to share what I have found generally for protein expression. Our lab has gotten into the habit of doing fresh transformation anytime that we need to do an expression. This seems to limit some of the variability from batch to batch and eliminates the concern of bacteria having been cured of their plasmid. As well, in one situation where degradation by cellular proteases (during protein expression) was a problem, I found that I could limit the in vivo degradation by performing inductions at 15C. To do this, I started the culture at 37, let it double and then dropped to 30C, after an hour or so dropped to 25C so on. Start with a low inoculum OD and keep in mind that this was in M9 (minimal medium). As well, I found that I could induce the culture at an OD600 of ~1.2 and let the induction go overnight (or 8h) and ended up with final OD's in the range of 2.7-3.0 which is great for minimal. I then took care of an in vitro proteolysis with a melange of protease inhibitors including some of those mentioned. I tried the BM tablets but found them too expensive for large scale work. I was then able to limit the protease inhibitors used in later steps of purification. Generally, at higher temps like 37 or 30, I've found that long inductions can lead to a higher level of inclusion body formation. All of our work has been in BL21(DE3) cells, so I can't really comment too much on bacterial strain variability. In the long run though, this protein expression stuff is a total crap shoot and one shouldn't give up too early. I think that I'm starting to freak out some of my co-workers as I'm talking about keeping my bugs happy and appear to be getting into bacterial karma...but hey, whatever works for you. Good luck, Randall C Willis, Researcher Biochemistry, Hosp for Sick Children 3522-555 University Ave Toronto, ON M5G 1X8 CANADA 416-813-5933 (ph) -5022 (fax) willis@gandalf.psf.sickkids.on.ca .