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, insert ed into pALTER and, after checking the nucleotide sequence, transferre d into pET81F1+. Procathepsin L, was expressed by induction of recombi nant E. coli strain BL21[DE3](pLysS) with IPTG and was found to be dep osited into inclusion bodies. These were isolated and solubilized in g uanidinium hydrochloride. The soluble proteins were sulphonated and pr ocathepsin L was obtained after gel filtration. Purified proenzyme was refolded by dialysis and autoactivated into a form of the expected si ze and enzymatic activity against a fluorogenic substrate.