Hi all, I am highly thankful to all of you for very useful replies. The following are the list of the replies to the query.
Query: Hi all, Sorry for a non-ccp4 query. I will highly appreciate your comments on the expression: I am trying to express HIS tagged ~60 kDa cytosolic protein from Bacillus in E.coli pET expression system. I tried codon+ strain, time of expression and various temperatures ( 18, 25, 30, 37C) for the expression. Neither full-length nor truncated form of the protein was found to be expressing (also checked by western blot). What other parameters should I explore to get the expression in Escherichia coli? Thank you very much for your comments. Amit K. Replies to above query: reply-1 Did you tried to check on the insoluble fraction to see if your protein is there? If the protein is insoluble tried the following: -Different E.coli strains -Less rich medium (LB instead of 2XYT) -Modify protein (shorter construct based on proteolysis, 2D structure prediction) - Most probably you will have to use a different vector using larger tags like GST, MBP, SUMO etc. - If all fails, you will need to go to other expression systems (Yeast, Pichia) reply-2 Looks like my very recent experience. In the expression profile, my protein started expressing after 20 hrs after low conc IPTG induction. I had to continue the expression for 36-40 hrs. reply-3 Are your codons optimized? I would look at the first 50 aa's to see if its particularly rich in rare codons, if so mutate them to more prefered e. coli codons. An N-His tag also helps with this since after about 50 aa's the ribosome doesn't tend to stall as much. The protein may also be toxic, so using a toxic resistance strain of e. coli is also good. Finally and maybe most simply I would get some fresh competent cells. If they aren't made properly they can have started to express the Lac inacativator protein which will absolutely kill your inducability. reply-4 You could try other tags, as His tags are not always the best. In addition, you didn't mention which side of the protein you tagged- it can make a difference whether it is N- or C-terminal. One last thing is to get the gene synthesized to codon optimize and RNA optimize. In this case I don't think codon optimization is a big factor, but your RNA could be forming interesting secondary structures which may inhibit translation in E. coli. reply-5 1. look up Bacillus megaterium expression system (commercial, pretty cheap & relatively easy). It's easier to use than some of the integration-based B. subtilis systems. Or you could ask around for a suitable B.sut. shuttle vector as an alternative. 2. Bacillus codon set is heavily biased towards AT-richness and even in the codon+ may not be expressed well - there may also be mRNA issues (stability). It's not hard to make synthetic genes these days - if you're desperate to continue using E. coli for this work you can make a synthetic codon-optimized version specific to the host. reply-6 We used a well-expressing soluble protein fusion (SUMO) at the N-term of a couple of B. anthracis proteins that we were trying to produce in E. coli, which improved the soluble yield of both targets tremendously. The disadvantage of a fusion is of course you produce a "non-native" protein- upon cleaving away the fusion (e.g. with thrombin or TEV), you will probably retain one or more exogenous residues. We picked SUMO because after cleaving with Ulp1 (SUMO protease) you produce a native protein with no additional residues from the fusion. If you want more details about this and things we tried to boost expression, please feel free to contact me off-list.
