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.
Dolinar
M
M
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Maganja
D B
DB
Turk
V
V
D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D004792:Enzyme Precursors / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D002403:Cathepsins; C063905:procathepsin L; D056668:Cathepsin L