His tag fusion question - (Oct/24/2007 )

Hi there,

I have a rather simple question: I wanted to express a protein with N-terminal His-tag using the pET28b+ vector. I didn`t remove the start codon from the gene sequence before I cloned it. Will this cause any trouble during expression?

Thanks, chalet2

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You do have a start codon at the beginning of the His tag, then it shouldnt be a problem.

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It might cause a problem depending on what you want to do with your portein.

For sure both proteins will be made but to different extent. E.coli do not strictly rely on the first ATG after the RBS. It is reported that also E.coli do use multiple ATGs shortly after the RBS.

So you will get a mixture of your protein with or without HIStag. But if you purify the HIStagged version later, it shouldn´t harm.

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