difficult expression of human gene in E. Coli - (Apr/12/2002 )
I have used pGEX4T2 and pET 30 to express fragments of a human gene in E.Coli.But it seems that they never worked for my gene.Rencently,I have substituted a new BL21 strain-BL21 CodonPlus RIL,in consideration that some rare condons to E.Coli used in my gene,but they still refused to work.By the way,the control(pGEX4T2 in BL21)expressed very well.Can anybody tell me some alternative methods to express the proteins or what's wrong with me?
Thank you very much!
I don't know you but there is probably nothing wrong with you. Don't take this E.coli business personally. This sort of expression is capricious at best and EVERY TRANSGENE must have its optimal conditions determined empirically. First try varying the temp and IPTG concentrations.
Human protein expression problems? Enter my world...
Many human proteins express poorly in E. coli. In my experience, the most common problem is that the proteins are expressed as insoluble or incorrectly folded soluble aggregates...the former is partitioned into inclusion bodies and can't be easily extracted in useful form, while the latter is often rapidly degraded by endogenous proteases. The best thing to try initially is expression overnight at ~25 degrees C, coupled with induction with a low concentration (as low as 0.1mM) IPTG.
Codon problems can be easily detected...just search the sequence for such codons and try E. coli strains with rare tRNA genes supplied.
Additionally, the protein may be toxic to the cells. In this case it helps to try expression in a strongly-repressing strain carrying the PLysS plasmid along with a T7lac promoter driven expression plasmid.
Welcome to my nightmares!
E. coli is a perfectly good organism for expressing genes from stupid, simple organisms that infect E. coli! Organisms that do not infect E. coli have no reason to have their system accustomed to the way E. coli cells function, therefore you have to try several different expression systems and see what works best (if any!).
I used a taged plasmid (GST tag) and RIL cells. Like you said, RIL cells substitute for the rare codons that are normaly absent in E. coli. In addition, the GST tag DOES HELP THE PROTEINS TO REMAIN SOLUBLE! Give the GST tag a try, it can be cleaved off by thrombin. I did manage to get very good expression using this tag! The plasmid it comes in is called pGEX-KG.
Also, like btavshan noted, the protein might be toxic to the cell, in which case try and use a very tightly-controlled expression system. T7 promoters are generally less leaky.
Good luck and be patient.