title: Expression and characterization of rat kallikrein-binding protein in Escherichia coli.
abstract: Rat kallikrein-binding protein is a novel serine-proteinase inhibitor that forms a covalent complex with tissue kallikrein. We have purified rat kallikrein-binding protein and cloned the cDNA and the gene encoding rat kallikrein-binding protein [Chao, Chai, Chen, Xiong, Chao, Woodley-Miller, Wang, Lu and Chao (1990) J. Biol. Chem. 265, 16394-16401; Chai, Ma, Murray, Chao and Chao (1991) J. Biol. Chem. 266, 16029-16036]. In the present study, we have expressed rat kallikrein-binding protein in Escherichia coli with a T7-polymerase/promoter expression system. A high level of expression was detected by an e.l.i.s.a. with an average of 24.2 mg of recombinant rat kallikrein-binding protein per 1 of culture. The recombinant protein appeared as a major protein in a crude extract of Escherichia coli on SDS/PAGE. It showed a molecular mass of 43 kDa and was recognized by polyclonal antibody to the native rat kallikrein-binding protein in Western-blot analysis. The recombinant rat kallikrein-binding protein has been purified to apparent homogeneity by DEAE-Sepharose CL-6B, hydroxyapatite Bio-Gel HPHT and Mono P 5/5 column chromatography. The purified recombinant rat kallikrein-binding protein showed immunological identity with the native rat kallikrein-binding protein purified from rat serum, in a specific e.l.i.s.a. To confirm the fidelity of the expression, the N-terminal ten amino acids of the recombinant rat kallikrein-binding protein were sequenced and were shown to match perfectly with those of the native rat kallikrein-binding protein. The purified recombinant rat kallikrein-binding protein formed SDS- and heat-stable complexes with rat tissue kallikrein (rK1) and T-kininogenase (rK10) in vitro, but not with other enzymes in the rat kallikrein gene family, such as tonin (rK2) and S3 protein (rK9), which indicates enzyme-specific binding. The properties of the recombinant rat kallikrein-binding protein including its size, charge, complex formation with target enzymes and immunological characteristics were compared with those of the native protein. This expression system provides a simple way to obtain a large amount of the biologically active recombinant protein, to study structure-function relationships of the rat kallikrein-binding protein and its interaction with its target enzymes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
authors.forename: J X
authors.initials: JX
authors.lastname: Ma
authors.forename: L
authors.initials: L
authors.lastname: Chao
authors.forename: G
authors.initials: G
authors.lastname: Zhou
authors.forename: J
authors.initials: J
authors.lastname: Chao
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D007610:Kallikreins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D015843:Serpins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D002352:Carrier Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015843:Serpins; C051324:kallistatin; D007610:Kallikreins