Hi here are few things to try for toxic proteins/genes supplement with glucose either eliminate overnight cultures or delay the destabilization of DE3 lysogens by including 0.5-1% glucose in the culture medium.
Another is to stabilize the toxic gene during induction. high cocnentration of antibiotic and replacing the medium twice prior to induction. A rough plan as follows Inoculate a single colony into 2mL TB+ 200ug/mL of antibiotic. grow cells at 37oc until OD600=0.2-0.6 collect cells (centrifuge 30secor so)remove sup and rsuspend in 2mL fresh media. Add a 100uL sample to 8mLTB+500ug/mL antibiotic and grow the culture at 37oC until OD600=0.2-0.6 collect cells (1000xg for 5min) and resuspend in fresh TB+500ug/ml antibiotic cantaining 1mM IPTG.incubate at 30oc for 2h before harvest. Note:The removal of medium removes the secreted beta-lactamase The above both suggestions are valid when stability of the plasmid is in question.It worked long back in certain situations of a plasmid instability. like earlier suggestions from Artem choice of promotor etc will be also crucial.other things like N-end rule,secondary site translational initation,secondary structure in mRNA transcript, instability of traget mRNA etc.these may not be the problem at all. I am guessing here. but before going for expensive gene synthesis you should definetly try Rosetta-gami strains becuase you know that your protein have unusual codon problems Just few thoughts, hope this helps. Padayatti PS On 1/10/07, Raji Edayathumangalam <[EMAIL PROTECTED]> wrote:
*** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** Hi Ailong, Hmmm... With MBP tagged, the total size of your expressed product is ~140 kDa. People in my lab regularly purify recombinant proteins upto 160-170 kDa but this can be tricky and need not be amenable in all cases. Here are some suggestions before you decide to make the synthetic gene: 1) Also try CodonPlus-RIL, CodonPlus-RPIL cells 2) Perhaps your protein product is toxic to E. coli. Try growing in autoinduction media (not LB) with overnight induction at lower temperatures (20-25C) without adding IPTG. Search for autoinduction info on the web. Also, look at papers by William Studier on the same. 3) Not sure if other tags like His tag, SUMO tag might help. 4) Sometimes, the plasmid or promoter driving expression needs to be changed 5) There are expression vectors for cold-shock expressions at ~15C HTH. Raji Ailong Ke wrote: > *** For details on how to be removed from this list visit the *** > *** CCP4 home page http://www.ccp4.ac.uk *** > > > Hi all, > > Here are some off-topic questions about protein expression that I would > appreciate some help with. > > I had a difficult time to express a 90 KDa human protein in E. coli. It > was fused to the C-terminus of MBP and contains a lot of rare arginine, > isoleucine and proline codons. The sequenced plasmid transforms well, but > the cells have difficulty to grow in large LB media, and no overexpression > were observed (I have yet to do a western). Trying BL21(DE)3RIL cells did > not help. I guess I need to try the Rosetta cells for the proline codons. > > My questions are: > 1) How big a protein can E. coli handle? Am I working at the borderline here? > > 2) I understand that rare codons cause premature termination and > degradation by tmRNA-mediated mechanism. But in reality, do I expect a > ladder of degradation bands or complete degradation? It is puzzling to me > not to see any MBP bands. The MBP vector expresses well. > > 3) One can fix the rare codon problem by providing a rare-codon tRNA > plasmid, or ordering a synthetic gene. Are there cases where the former > approach failed, but the latter one succeeded. > > 4) Can anyone recommend an on-line server to do codon/RNA secondary > structure/GCcontent optimization? > > 4) Can anyone recommend a reliable company for gene synthesis, with > reasonable $/bp pricing? > > > > > -- Raji Edayathumangalam Postdoctoral Fellow The Rockefeller University Box 224. 1230 York Avenue New York, NY 10021
-- Pius S Padayatti Department of Biochemistry, Structural Biology Division, School of Medicine, RT-500 Case Western Reserve University, Cleveland, Ohio-44106, 216-368-6833
