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 mitochondr ial ubiquinol-cytochrome c reductase was cloned and sequenced. This cD NA 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 am ino acid sequence of QPc-9.5 kDa deduced from this nucleotide sequence is the same as that obtained by protein sequencing except that residu e 61 is tryptophan instead of cysteine, The QPc-9.5 kDa was overexpres sed in Escherichia coli JM109 cells as a glutathione S-transferase fus ion protein (GST-QPc) using the expression vector, pGEX/QPc, The yield of soluble active recombinant GST-QPc fusion protein depends on the i nduction growth time, temperature, and medium. Maximum yield of recomb inant fusion protein was obtained hom cells harvested 3 h postinductio n of growth at 27 degrees C on LB medium containing betaine and sorbit ol, QPc-9.5 kDa was released from the fusion protein by proteolytic cl eavage with thrombin. Isolated recombinant QPc-9.5 kDa showed one prot ein band in SDS-polyacrylamide gel electrophroesis corresponding to su bunit VII of mitochondrial ubiquinol-cytochrome c reductase. Although the isolated recombinant QPc-9.5 kDa is soluble in aqueous solution, i t is in a highly aggregated form, with an apparent molecular mass of o ver 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 titrat ion of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that the reco mbinant protein may be in the functionally active state.