I have a protein characterising and it happens to be a GPCR from bioinformatic analyses of the Class A. I have the gene coding for it in pET 15b vector and have tried to express it by IPTG induction to no avail. Can anyone please suggest a way of going about this problem?
Do you have a positive control for your expression experiment? Ideally, something cloned on pET15b that expresses well under your conditions.
Have you sequenced your final construct? Are you sure that your sequence was inserted in frame? Besides, can you exclude the introduction of additional stop codons?
First thing to check would be the sequence of the construct. I have been burned several times with supposedly "sequence-verified" plasmids even from reputable vendors. As some of the other pointed out above, GPCRs are usually not highly expressed. Have you tried to do an RNA synthesis to see if the gene is expressing at the mRNA level?
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