Over-expression and purification of active serine proteases and their variants from Escherichia coli inclusion bodies

This unit describes the over-expression and purification of active serine proteases and their variants from E. coli inclusion bodies. The strategy includes the folding and purification of a stable zymogen precursor protein, and its later activation with the appropriate convertase to the less stable but active protease. A test to follow the presence of activity in the samples, together with an active-site titration protocol to determine the number of active sites per mole of total protein are provided. It should be emphasized that although most of the protocols described are applied to a specific example, they are fairly representative of the methods and approaches generally used for laboratory-scale preparation of other recombinant serine proteases. The critical steps and how this template protocol can be adapted for the purification of other serine proteases are described.