amino sequence of expressed protein - doesn't match coding sequence (Nov/05/2001 )
I constructed a novel E.coli secretory expression system and expressed a peptide. The target protein was directed into the medium by activity assaying and the signalpeptide was removed exactly from the target peptide by N-terminal amino acid sequencing of the purified expressed product. However,to my surprise the N-terminal 15 amino sequence of the expressed target protein doesn't correspond totally with its coding gene? there are two alteration: the 5th amino acid (its coding sequence is GAC) ASP was altered into ASN; the 12th amino acid ASN(its coding sequence is AAC) was altered into ASP. Can any insightful one give me an explanation?
In both cases it appear that only one nt was changed, which might be caused by using low-fidelity theromsotable polymerase during PCR propagation. However I would double-check the sequences of both original construct and that of expressed protein. You must make sure that the anomalities do not arise from the sequences you obtained. Have a good luck.