Endoproteolytic Pattern Observed During Refolding of a Human Exopeptidase Proenzyme, Procathepsin H, Produced in Escherichia coli

Marko Dolinar*, Andreja Mehle1, Bojana Mozetič Francky2, Ana Schweiger and Vito Turk

Ljubljana Pharmacies, Ul. stare pravde 11, SI-1000 Ljubljana

2Biohit d.o.o., Gorazdova 7, SI-1000 Ljubljana
 Department of Biochemistry and Molecular Biology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia

Article history:

Received November 11, 1999
Accepted January 21, 2000

Key words:

cathepsin H, endopeptidase, exopeptidase, 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 BL21DE3pT-Trx. The majority of the recombinant procathepsin H was present in the insoluble form, which comprised 25 % of total bacterial proteins. After solubilisation of inclusion bodies, procathepsin H was refolded by dialysis. In the process of renaturation, recombinant procathepsin H was proteolytically degraded into several distinct fragments which were detected by monoclonal antibodies directed toward the N- or C- terminus of the mature enzyme. The degradation pattern was typical for an endopeptidase and a stable LMW fragment of 14 kDa could be assigned to the C-terminal region of the mature enzyme. In refolding, human procathepsin H thus undergoes autolysis by an endopeptidase mechanism, previously disputed for this enzyme. 

*Corresponding author:           Marko.Dolinar@IJS.SI
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