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Including "M" and "STOP" codons in my insert

Recombinant proteins

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#1 IPI

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Posted 19 August 2012 - 10:21 PM

Hi There,

I intend to overexpress a fusion recombinant polypeptide in L. lactis, consisted of two partial proteins connected by a linker. Since the start and the stop codons are not included in this insert, I was wondering, should I design an "M" and a "STOP" codon at the beginning and end of the insert fragment, respectively?

By the way, my expression vector does not provide an "M" upstream of the insert and the stop codon of the vector actually stops at 6 amino-acids (i.e. SSSSFL) after the C-terminal of my intended fusion polypeptide.

With many thanks in advance,

P.S.

Supplemental Info:
===================

Host: Lactococcus lactis strain NZ3900
Vector: pNZ8149
From NIZO (NICE® Expression System for Lactococcus lactis)

#2 ascacioc

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Posted 20 August 2012 - 10:13 AM

Your construct inside the vector should be promoter-rbs-ATG-protein1-linker-protein2-stop codon-terminator in order to have the protein expressed. If the vector is missing any of these, you should insert them together with the insert construct.

Andreea

#3 IPI

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Posted 20 August 2012 - 08:09 PM

Thank you for your response Enthusiast.







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