PacI affect protein translation? - (Oct/06/2011 )

Hi All

I have some protein expression issue recently, and hope some can give some suggestions. I try to over express one protein from GPD promoter containing vector. There is only one restriction site that can be used for cloning-- PacI. This vector was first designed and made by our previous lab member, but has not been tested. I cloned my gene at Pac1 site and correct orientation of the gene was confirmed by colony PCR and restriction digestions. Transformed into the cells and did western with no protein detected. I then did RT-PCR to confirm the expression of the gene, and I got correct size band. It looks like that the gene is expressed but not get translated. I also try to over express Dsred using that vector but I did not see red fluorescent under the microscopy.

Here comes a question? Does anyone think that by putting a stop codon in front of the start codon of a gene affects protein translation, but not gene transcription? (PacI site = TTAATTAA). Has anyone used Pac1 site for over expression of protein?

Thanks for sharing your opinions.

all the best,

Chen

Have you sequenced the plasmid upstream of the PacI site? If a start codon is present upstream that is out of frame with your gene or in frame with the stop codons, that will be a big problem. I would make a habit out of sequencing any DNA that I get from somebody else, no matter how much I trust them (except vectors purchased from companies, i'm not that obsessive). I have had multiple times where sequences that other people meant to put into a vector, were never verified. That's my best guess for solving your problem.

The only other thing I can think of is your translational start site context. Did you put a kozak's sequence upstream of your start codon? In some cases a very bad start codon context and dramatically reduce protein expression.

Best of Luck.