Human F1-ATPase: Molecular Cloning of cDNA for the Beta Subunit
1986 年 99 巻 1 号 p. 135-141
詳細
1986 年 99 巻 1 号 p. 135-141
F1-ATPase is the major enzyme for ATP synthesis, and its β subunit is the catalytic site. To date, no full-length cDNA for the eukaryotic F1 gene has been reported. Human F1 was studied because of its importance in medicine and cell biology. Here we report molecular cloning of a full-length cDNA for the human F1β subunit and purification of the human F1β subunit. The HeLa cell cDNA library constructed in an expression vector λgt11 was screened with antiserum against the yeast F1β subunit. One of the positive phage DNAs containing the human F1β gene and its flanking regions (1.8 kilobase pairs) was sequenced by the dideoxy chain termination method. The open reading frame started from a putative signal presequence, which was rich in both serine and arginine. There was a homologous segment in the signal presequence of human ornithine transcarbamoylase and that of F1β. The precursor of F1β was expressed in E. coli harboring a plasmid which had been constructed with T5 promotor and the F1β cDNA. Both the precursor and mature form of F1β were detected in HeLa cells in a pulse-chase experiment. The amino acid sequence of 480 residues (51, 568.3 daltons) following the presequence was highly homologous with that of mature beef heart F1β (97.5%) and E. coli Flβ (71.7 %), but the codon usage in the human gene was very different from those of reported genes coding for F1β of other species.
