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Expression in Escherichia coli and characterization of the fatty-acid-binding protein from human muscle.

ABSTRACT: The coding part of the cDNA encoding human muscle fatty-acid-binding protein (FABP) was ligated in the pET8c vector and expressed under the control of the lacUV5 promoter. After induction with isopropyl beta-D-thiogalactopyranoside, almost 12% of the cytoplasmic proteins consisted of FABP. The protein could be isolated after sonication of the bacterial pellet followed by (NH4)2SO4 precipitations, anion-exchange chromatography and gel filtration. The muscle FABP produced in Escherichia coli has an isoelectric point of 5.3 and is recognized by anti-(human muscle FABP) antiserum after Western blotting. The purified FABP has a preference for binding to palmitic acid and C18-C22 (poly)unsaturated fatty acids, and no affinity to palmitoyl-CoA or other hydrophobic ligands tested. The dissociation constant for oleic acid is 0.58 microM, with a binding stoichiometry of 0.72 mol of fatty acid/mol of protein. The physicochemical and binding characteristics of the protein were in complete agreement with those of FABP isolated from human skeletal muscle.

SUBMITTER: Peeters RA

PROVIDER: S-EPMC1151349 | biostudies-other | 1991 Sep

REPOSITORIES: biostudies-other

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Project description: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.

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Expression in Escherichia coli and characterization of the fatty-acid-binding protein from human muscle.

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