I induced two cultures at 0.5 OD600: 1 with just the vector, and another with my ORF in the vector. However, following induction with 1mM IPTG with shaking at 140 rpm at 37 degree C for even 6 hours did not induce protein overexpression, even in the culture harboring just the vector. The vector and the clone are both proper (as confirmed by sequencing, digestion profile, PCR screening).
Help and suggestions would be appreciated.
That is common phenomenon for protein expression. Protein expression depends on many factors: Temperature, IPTG concentrations, the fresh of used compettent cells...You have to try to optimize them first....
Other thing you can do is checking your amino acid sequence whether is belong to " rare codon"? or not. Sometime, protein expression in other host strains will change, for example " Rossetta instead of BL21(DE3).
I agree protein expression depends on several factors. You can refer the paper on optimization of protein expression conditions in E. coli. Here is the link to access the paper.
https://www.researchgate.net/publication/5603846_High-level_expression_of_soluble_viral_structural_protein_in_Escherichia_coli
Article High-level expression of soluble viral structural protein in...
If your empty vector does not express GST it means there is problem in your Vector backbone, IPTG or strain. Go one by one. First, change the IPTG stock. If it does not work, check your vector backbone and purify it from newly transformed fresh competent cells. You can also opt for other strains suggested above. Hope this will solve your problem at initial stage.
Use BL21-codonplus (DE3) -RIL strain as your competent cells. After the induction grow it at lower temperature like 25C overnight. But you need to optimize this temperature
Thank you all. The latest update is that my Vector gives a good GST band after 1 hr, till 8 hrs at 37 degrees, but my clone doesnt. Although, the clone is perfectly in frame. It would have been nice if I could have been able to procure Rosetta or RIL or pLysS strains.
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