proteolytic cleavage - proteolytic cleavage (Jul/10/2008 )

Hello,

I am trying to express a protein in sf9 cells, using a gateway vector which has a histag, but the protein undergoes proteolytic clevage during expression and I get very little amount of the protein, plus the protein is a dimer, hence unless it is expressed in the cells and present for sometime it will not form the dimer

I analysed the amino acid sequence for proteolytic cleavage enzymes and am uploading the file, would be happy if someone could suggest me how to go about expressing the protein without lossing it to the proteolytic enzymes.


thekid

If proteolysis occurs only after expression during / after lysis use protease inhibitors during purification (e.g. complete tablets from Roche Applied Science). If it happens intracellular you can try to clone them to be secreted or use another cell line.
Maybe you can increase your transfection efficiency with FuGene HD so that you get higher levels of protein expression (and less cytotoxic effects which induce proteolysis) and you can harvest the cells earlier before proteolysis occurs. However do the optimization titration described in the pack insert.

Sorry for having no better suggestions...