I have cloned the E2 gene of JEV in BamHI and HindIII sites of pET28a vector along with start codon ATG. Tried to induce the protein with several concentrations of IPTG ranging from 0.2 to 1 mM, temperatures 25, 30 and 37 degree celsius and also used different competent cells like BL21(DE3), pLyss and C41. But the protein is not getting induced and no bands are observed in western blots using Anti-His antibody. Please suggest some solutions.
Induction conditions are also critical along with concentrations. please indicate the induction temperatures you used
E. coli might not be the right choice for a viral glycoprotein. These things tend to depend on glycosylation, and as you will understand, E. coli does not glycosylate. You could try the MBP fusion strategy to get the initiation rate up and improve folding. But more likely than not you'll still see misfolding, lack of activity and proteolytic degradation with this kind of glycoprotein. This should be done in mammalian cells.
It might help to use Rosetta competent cells that have tRNAs for codons that are rare in E. coli.
http://www.emdmillipore.com/US/en/product/Rosetta%E2%84%A2%28DE3%29pLysS-Competent-Cells---Novagen,EMD_BIO-70956
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