In B.subtilis spore surface display cot partner with native promoter is fused in frame with a passenger protein via a linker like GGGGS. The assumption here is that a fusion mRNA is formed, and translation starts from the cot partner START codon and ends at the stop CODON of the passenger protein. The linker permits the independent folding of the two proteins. My question is will omitting the N terminal START codon of the passenger protein (enzymes in my case) affect its stability and activity? I'm having multiple enzymes fail with multiple cot partners like cotB,CotC and CotG. I have not included the START codon in the passenger protein in my constructs. I have been checking the literature and so far in every study they have included the start codon of the passenger protein. Any thoughts about its impact?
The answer is that it depends. Most often the N-terminal Met is not needed in the protein, but there are some cases where it is. So I would think that while usually you would not need to include it, but on the other hand there is no strong reason not to include.
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