Hi
I have purified His-tagged protein. But after SDS-PAGE analysis I found a lot of protein seem to lost His-tag that is located at N-terminus and a lot of protein of interest might be lost at Ni-NTA purification step. SDS-PAGE analysis using ImageJ showed that the His-tag is cleaved between His-tag and protein of interest. So I want to replace the linker between His-tag and protein of interest using site specific mutagenesis. Any good advice about good linker that is resistant proteases from E. Coli?
Hi there,
How sure are you that the protein degradation you spot actually occurs on the Nter? Did you perform WB with antiHis to show the degradation product does not contain His tag anymore? The band shift you observe looks bigger than just a few aa (which should be the case is cleavage in the thrombin site...). About alternative linker, 10 aa linker (Gly4Ser)2 is usually used as a flexible spacer.
Hi Dominique,
I confirmed the absence of his-tag by His-tag specific in-gel stain instead of WB. And I compared the SDS-PAGE band pattern with that of GST-fusion one and others. I could not see lower band on other gels thus I thought that this is His-tag cleaved one. I also made calibration curve using the bands of markers and the size difference of two bands are 1.9 kDa. I apologize you not to add extra information about the amino acid sequence because I forgot to show the TEE sequence.
To me it looks like the result of some protease activity. Have you tried to load your sample with protease inhibitor cocktail?
Yes, I added EDTA free protease inhibitor cocktail. I do want to know which protease cut the seqence to substitude the amino acid by site specific mutagenesi.
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