I have cloned a gene encoding an enzyme from gram positive bacteria in E.coli BL21(DE3), tried all the IPTG conc. and temperatures but there is no sign of expression. Is there any incompatibility of signal sequence of the gene in E.coli? Please Help!!
you may try to use different combinations of vectors and BL21 strains. Expression recombinant proteins by using vectors such as pGEX or pMAl with N termnial fusion tags could improve the expession. In addition, check if there are rare codons in the enzyme that prevent expression in E.coli. If the enzyme has a signal peptide，it may be secreted into specific cell compartments or outside the cell. in that case, the whole cell culture medium should be used for SDS-PAGE.
Hi there,
For the desperate cases auto induction medium may be the solution...
Hello Sebastian Schmitt
Sir,The sequence of the construct is fine. Please clarify 'what kind of signal sequence' means? I am using pET28a vector for the expression. No sign of expression means there is no difference between the empty vector, uninduced control and the induced cultures and that's why I have not proceeded for the western blot. Also I have tried all the possible conditions but nothing worked. Should I need to remove the signal sequence from the gene and then clone it in pET?
Thank You.
Thank you Ling Tong Liu for the suggestions, I am using pET28a vector in BL21 (DE3) strain. How to check for rare codons?
Hi Namrata,
The following rare codon analysis tools
may be helpful.
https://www.genscript.com/tools/rare-codon-analysis#opennewwindow.
To increase the chances of sucessful expression, I also recommend BL21- rosetta or BL21-CodonPlus. These strains contain extra copies of genes code for the tRNAs that most frequently limit translation of foreign proteins in E.Coli.
Thank you Ling Tong Liu , will try a different host like you suggested.
Hello,
Now my protein is expressing but in the pellet. Can I proceed for purification or do I try to express it in soluble form?
Thank You.
Hi there,
If you want to recover your protein from the pellet, you have first to solubilise it into chaotropic containing buffer (6M GdmCl or 8M urea) and then to perform purification/renaturation process which is not so easy. Instead I would try to get something soluble/native from the start even if it is not 100%. The fact that you actually express the protein in inclusion bodies is a good starting point as inclusion bodies come from aggregation due to protein misfolding. By slowing down the expression (lowering IPTG, lowering temperature, coexpressing chaperones, using autoinduction media) you should be able to get some soluble protein which is much easier to handle through purification steps.
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