Denaturing the Fusion Protein

Occasionally, the factor Xa site of a particular fusion is inaccessible to the protease, owing to the three-dimensional structure of the protein. In this case, denaturing the protein can sometimes allow the factor Xa to cleave (see Note 18).

1. Either dialyze the fusion against at least 10 volumes 20 mM Tns-HCl, 6M guani-dine hydrochloride, pH 7 4 for 4 h, or add guanidine hydrochloride directly to the sample to give a final concentration of 6M

2. Dialyze against 100 volumes column buffer, 2 times at 4 h each

During refolding, one has to balance between two objectives. For factor Xa to cleave it must be present before the protein has completely refolded, so removing the denaturant quickly is desirable. However, when the denaturant is removed quickly, some proteins will fail to refold properly and precipitate. Stepwise dialysis against buffer containing decreasing amounts of guanidine hydrochloride can prevent precipitation of the fusion protein; halving the guanidine concentration at each step is convenient, but cases where 0 1M steps are necessary have been reported. However, if the fusion protein is able to refold into a factor Xa-resistant conformation, it may be better to dialyze away the denaturant in one step and take the loss from precipitation in order to maximize the amount of cleavable fusion protem recovered.

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