RECOMBINANT BOVINE HEART MITOCHONDRIAL F1-ATPASE INHIBITOR PROTEIN - OVERPRODUCTION IN ESCHERICHIA-COLI, PURIFICATION, AND STRUCTURAL STUDIES

A synthetic gene coding for the inhibitor protein of bovine heart mito chondrial F1 adenosine triphosphatase was designed and cloned in Esche richia coli. Recombinant F1-ATPase inhibitor protein was overproduced in E. coli and secreted to the periplasmic space. Biologically active recombinant F1-ATPase inhibitor protein was recovered from the bacteri al cells by osmotic shock and was purified to near homogeneity in a si ngle cation-exchange chromatography step. The recombinant inhibitor pr otein was shown to inhibit bovine mitochondrial F1-ATPase in a pH-depe ndent manner, as well as Saccharomyces cerevisiae mitochondrial F1-ATP ase. Thorough analysis of the amino acid sequence revealed a potential coiled-coil structure for the C-terminal portion of the protein. Expe rimental evidence obtained by circular dichroism analyses supports thi s prediction and suggests F1I to be a highly stable, mainly alpha-heli cal protein which displays C-terminal alpha-helical coiled-coil interm olecular interaction.