Purification and characterization of recombinant tissue kallikrein from Escherichia coli and yeast.
A full-length rat tissue kallikrein cDNA was constructed by oligonucleotide engineering through an extension of RSK 1105, a partial cDNA clone containing 534 bp of the 3' end of tissue kallikrein, followed by site-directed mutagenesis to remove the vector sequence from within the chimaeric coding sequence. The cDNA has been cloned both into the plasmid pET3b under the control of the T7 promoter/polymerase system, and into the shuttle vector PYE directed by the alpha-factor promoter. Expression in Escherichia coli was detected by direct radioimmunoassay, and recombinant kallikrein of 36 kDa was identified by Western-blot analysis using both polyclonal and monoclonal antibodies to rat tissue kallikrein, and by autoradiography of 14C-labelled L-amino acid-labelled-protein synthesis in the presence of rifampicin. Expression in yeast was also detected by direct radioimmunoassay, and recombinant kallikrein was identified by Western-blot analysis with a molecular mass of 39 kDa. The recombinant kallikrein from yeast, however, remained mostly inactive. Kallikrein was purified to apparent homogeneity from E. coli by DEAE-Sepharose CL-6B and aprotinin-affinity column chromatography and confirmed by the N-terminal ten-amino-acid sequence, which matched the deduced sequence from the cDNA. Both E. coli and yeast recombinant kallikreins have Tos-Arg-OMe-esterolytic and kininogenase activities similar to those of purified tissue kallikrein. Comparisons were made between recombinant kallikreins and rat tissue kallikrein with respect to size, charge, substrate specificity, susceptibility to inhibitors and immunological properties. Our results open the way for the study of kallikrein structure-function relationships through protein engineering.
Wang
J
J
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
Chao
J
J
Chao
L
L
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D003902:Detergents; D004926:Escherichia coli / Q000235:genetics*; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007610:Kallikreins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D020840:Tissue Kallikreins
D003902:Detergents; D011994:Recombinant Proteins; D007610:Kallikreins; D020840:Tissue Kallikreins