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?
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amino sequence of expressed protein
1 reply to this topic
Posted 07 November 2001 - 10:00 PM
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.