I am using the pET-SUMO cloning system from ThermoFisher to recombinantly produce proteins in E. coli BL21(DE3) cells and have found success with several proteins expressed by the gram-negative bacterium, Brucella. However, I am planning to proceed with the cloning and expression process for an additional protein whose sequence begins with the alternative start codon GTG instead of the typical ATG. Is this something that could affect recombinant fusion protein production? Or should I design my primers to begin with ATG instead? Thanks!
I would take no chance and replace the GTG start codon by an ATG codon using appropriate primer.
It might also be worthwhile to run PsiBLAST on your sequences to see what other constructs exist in other species. Sometimes the gene is misannotated and an alternative start site gives you the proper construct.
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