Hello everyone and thanks for reading his question.
I found during bacterial protein production that I had set the centrifuge much higher than intended when pelleting intact bacteria.
My target protein was GFP tagged. Following the pelleting step, the bacterial pellet was perfectly green and the supernatant was absolutely clear.
I performed my usual protocol for purification and the target protein was badly cleaved. I have no way to make comparison because I just made this construct and this is the first attempt for protein purification.
My question is whether or not spinning the intact bacteria at too great a force can trigger something in the bacteria that results in enhanced proteolytic cleavage?
Spinning intact bacteria likely had little impact on the proteins. The most likely reason is lack of sufficient protease inhibitors. What steps did you do after pelleting your cells? If you run out a sample of your bacterial pellet on a gel do you see the predicted size fusion protein?
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