Answers: 1. Large. Although I cannot think of good examples right now, I think that several hundred kD should be fine. 2. Good question. Is your protein partially expressed when the degradation takes place, or not at all? If it is partially expressed, would you 'see' it or would it be targeted for immediate (complete) degradation (because it is recognized as 'faulty') or would it become insoluble, or... ? 3. We have had at least one project in the lab for which all approaches failed until we ordered a synthetic gene. (Question 4 skipped, not omitted by mistake :-) 5. We have used GeneArt (http://www.geneart.com/) and have been very happy with their service. I think their price is roughly $1 per bp, so a 500 amino acid protein costs approximately $1500. It sounds like a lot of money until you convert the price to salary and calculate the number of hours you can bang your head against the wall for that money. They will do codon optimization and other things you might want (like inserting restriction sites in your sequence so you can later cut out certain domains) and then they will generate the gene in the plasmid of your choice. From what I recall, the manufacturing time is a couple (3?) weeks. So far (for us) they delivered as promised. Their web site has lots of information. Hope this helps. Mark -----Original Message----- From: [EMAIL PROTECTED] To: ccp4bb@dl.ac.uk Sent: Wed, 10 Jan 2007 10:10 AM Subject: [ccp4bb]: Off topic: protein expression problem *** 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? ________________________________________________________________________ Check Out the new free AIM(R) Mail -- 2 GB of storage and industry-leading spam and email virus protection.
