REQUIREMENT OF PRO-PEPTIDE IN PROPER FOLDING OF SUBTILISIN-LIKE SERINE PROTEASE TK0076

Subtilisin-like proteases are characterized by a catalytic triad of the three amino acids Asp His and Ser. Tk0076 is a subtilisin-like serine protease originated from Thermococcus kodakaraensis. Regions corresponding to signal-peptide, propeptide and the mature protein were predicted by homology modeling. Homology comparison revealed that Asp 215 , His 247 and Ser 424 constitute the catalytic triad of the protein. Gene encoding Tk0076 was cloned and expressed in Escherichia coli. The protein was produced in the soluble form when the gene contained the sequence corresponding to the pro-peptide whereas it was produced in the insoluble form without the sequence corresponding to pro-peptide under the same expression system. Attempts to refold the protein properly in the absence of pro-peptide were unsuccessful indicating that pro-peptide is essential for proper folding of Tk0076.