Hi,
I have cloned some three different constructs which are putative membrane protein of Mycobacterium tuberculosis into pET28a with N-term His tag. I tried to express them in BL21, Rosetta, C41, C43 strains of E.coli but it failed to express in soluble or insoluble fraction.
Is there any other E.coli strain that I should try before switching to Mycobacterium smegmatis?
Hi,
Your desired genes are in the frame in the constrcut ? If its fine with the frame still you could not see the expression try to check the expression vector which has gst or other tag which can increase the solubility of your protein.
Dear Haaris,
Before thinking of changing the E .coli strain, try to troubleshot the problem. In fact, expression of heterologous proteins in E. coli is a challenging task and may affects the viability of the host cell. We had similar problem and the solution came from considering several factors such as the type of promoter, the plasmid and the temperature for growing the cells...
I would strongly recommend you to study the useful recommendations in this link:
https://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/index.html
Good luck!
Hi Haaris,
Based on my previous experience in expressing proteins, I think you can try different truncations and different expression vectors, such as pACYC, which my colleagues in the lab used to express prokaryotic transmembrane proteins well. GST or MBP tags can improve protein solubility.
Good luck!
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