Cloning, gene sequencing, and expression of the small molecular mass ubiquinone-binding protein of mitochondrial ubiquinol-cytochrome c reductase.
The cDNA encoding QPc-9.5 kDa (subunit VII) of bovine heart mitochondrial ubiquinol-cytochrome c reductase was cloned and sequenced. This cDNA is 665 base pairs long with an open reading frame of 246 base pairs that encodes an 81-amino acid mature QPc-9.5 kDa. The insert contains 395 base pairs of a 3'-noncoding sequence with a poly(A) tail. The amino acid sequence of QPc-9.5 kDa deduced from this nucleotide sequence is the same as that obtained by protein sequencing except that residue 61 is tryptophan instead of cysteine. The QPc-9.5 kDa was overexpressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-QPc) using the expression vector, pGEX/QPc. The yield of soluble active recombinant GST-QPc fusion protein depends on the induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth at 27 degrees C on LB medium containing betaine and sorbitol. QPc-9.5 kDa was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPc-9.5 kDa showed one protein band in SDS-polyacrylamide gel electrophroesis corresponding to subunit VII of mitochondrial ubiquinol-cytochrome c reductase. Although the isolated recombinant QPc-9.5 kDa is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1 million. Addition of detergent deaggreates the isolated protein to the monomeric state, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. The recombinant QPc-9.5 kDa binds ubiquinone and shows a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that the recombinant protein may be in the functionally active state.
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Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078, USA.
Deng
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Yu
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics; D014450:Electron Transport Complex III / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D013056:Spectrophotometry, Ultraviolet; D014451:Ubiquinone / Q000378:metabolism*
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014451:Ubiquinone; D014450:Electron Transport Complex III