Endoproteolytic pattern observed during refolding of a human exopeptidase proenzyme, procathepsin H, produced in Escherichia coli

Cathepsin H is a lysosomal cysteine proteinase with exopeptidase activity. Total mRNA was purified from human endometrium cells and converted to cDNA. The procathepsin H coding region was amplified by specific primers, cloned and expressed by the T7-polymerase controlled expression vector pET3a and the bacterial strain Escherichia coli BL21[DE3]pT-Trx. The majority of the recombinant procathepsin H was present in the insoluble form, which compris ed 25 % of total bacterial proteins. After solubilisation of inclusion bodi es, procathepsin H was refolded by dialysis. In the process of renaturation , recombinant procathepsin H was proteolytically degraded into several dist inct fragments which were detected by monoclonal antibodies directed toward the N- or C- terminus of the mature enzyme. The degradation pattern was ty pical for an endopeptidase and a stable LMW fragment of 14 kDa could be ass igned to the C-terminal region of the mature enzyme. In refolding, human pr ocathepsin H thus undergoes autolysis by an endopeptidase mechanism, previo usly disputed for this enzyme.