EXPRESSION AND CHARACTERIZATION OF RAT KALLIKREIN-BINDING PROTEIN IN ESCHERICHIA-COLI

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 encod ing rat kallikrein-binding protein [Chao. Chai, Chen, Xiong, Chao, Woo dley-Miller, Wang, Lu and Chao (1990) J. Biol. Chem. 265, 16394-16401; Chai, Ma, Murray, Chao and Chao (1991) J. Biol. Chem. 266, 16029-1603 6]. In the present study, we have expressed rat kallikrein-binding pro tein in Escherichia coli with a T7-polymerase/promoter expression syst em. A high level of expression was detected by an e.l.i.s.a. with an a verage of 24.2 mg of recombinant rat kallikrein-binding protein per 1 of culture. The recombinant protein appeared as a major protein in a c rude extract of Escherichia coli on SDS/PAGE. It showed a molecular ma ss of 43 kDa and was recognized by polyclonal antibody to the native r at kallikrein-binding protein in Western-blot analysis. The recombinan t rat kallikrein-binding protein has been purified to apparent homogen eity by DEAE-Sepharose CL-6B, hydroxyapatite Bio-Gel HPHT and Mono P 5 15 column chromatography. The purified recombinant rat kallikrein-bind ing protein showed immunological identity with the native rat kallikre in-binding protein purified from rat serum, in a specific e.l.i.s.a. T o confirm the fidelity of the expression, the N-terminal ten amino aci ds of the recombinant rat kallikrein-binding protein were sequenced an d were shown to match perfectly with those of the native rat kallikrei n-binding protein. The purified recombinant rat kallikrein-binding pro tein formed SDS- and heat-stable complexes with rat tissue kallikrein (rK1) and T-kininogenase (rK10) in vitro. but not with other enzymes i n the rat kallikrein gene family, such as tonin (rK2) and S3 protein ( rK9), which indicates enzyme-specific binding. The properties of the r ecombinant rat kallikrein-binding protein including its size, charge, complex formation with target enzymes and immunological characteristic s were compared with those of the native protein. This expression syst em 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 tar get enzymes.