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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:
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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
