Abstract
The propeptide of subtilisin, an alkaline serine protease, is known to be required for the folding of subtilisin, functioning as an intramolecular chaperone. Upon folding of prosubtilisin, the propeptide of 77 amino acid residues is autocatalytically cleaved. A histidine-tag was added to the N-terminal end of prothiolsubtilisin E, or prosubtilisin(S221C), in which the active site serine residue at position 221 was substituted with cysteine. The histidine-tagged prosubtilisin(S221C) was denatured and immobilized on Ni-NTA resin. The denatured protein was then refolded on the resin, and the efficiency of the renaturation was determined by the efficiency of the propeptide cleavage. It was found that the cleavage of the propeptide was independent of the concentration of prosubtilisin(S221C), indicating that the autoprocessing is an intramolecular reaction. We also showed that prosubtilisin(S221A) can be autoprocessed if it is mixed with histidine-tagged prosubtilisin(S221C). These results demonstrate that prosubtilisin is intrinsically capable of being autoprocessed in an intramolecular manner, while it can also be processed in an intermolecular manner if it exists at higher concentrations.
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