Expression of full-length human procathepsin L cDNA in Escherichia coli and refolding of the expression product.

From human embrional lung fibroblasts mRNA was obtained and converted to cDNA. The procathepsin L coding region was amplified by PCR, inserted into pALTER and, after checking the nucleotide sequence, transferred into pET81F1+. Procathepsin L was expressed by induction of recombinant E. coli strain BL21[DE3](pLysS) with IPTG and was found to be deposited into inclusion bodies. These were isolated and solubilized in guanidinium hydrochloride. The soluble proteins were sulphonated and procathepsin L was obtained after gel filtration. Purified proenzyme was refolded by dialysis and autoactivated into a form of the expected size and enzymatic activity against a fluorogenic substrate.