Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli.
A cDNA clone encoding the human cysteine protease cathepsin L was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at alkaline pH. 38-kDa procathepsin L was purified by gel filtration at pH 8.0, and a 29-kDa form of the enzyme was purified by gel filtration after autoprocessing of the proenzyme at pH 6.5. The kinetic properties of the 29-kDa species of recombinant cathepsin L were similar to those published for the human liver enzyme (Mason, R. W., Green, G. D. J., and Barrett, A.J. (1985) Biochem. J. 226, 233-241), using benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide as substrate. However, the stability of the recombinant enzyme, and its pH optimum for this substrate was shifted to a higher pH. Structure-function studies of cathepsin L were performed by constructing mutations in either the propeptide portion or the carboxyl-terminal light chain portion of the protein. These constructions were expressed in the E. coli system, and enzymatic activities were assayed following solubilization, renaturation, and gel filtration chromatography of the mutated proteins. Deletions of increasing size in the propeptide resulted in large proportional losses of activity, indicating that the propeptide is essential for proper enzyme folding and/or processing in this renaturation system. Deletion of part of the light chain containing a disulfide-forming cysteine residue or a single amino acid substitution of alanine for this cysteine residue resulted in almost complete loss of activity. These data suggest that the disulfide bond joining the heavy and light chains of cathepsin L is essential for enzymatic activity.
Smith
S M
SM
Howard Hughes Medical Institute, National Cancer Institute, Bethesda, Maryland 20892.
Gottesman
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005796:Genes; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014462:Ultrafiltration
D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L