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 mitochondrial F1 adenosine triphosphatase was designed and cloned in Escherichia 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 bacterial cells by osmotic shock and was purified to near homogeneity in a single cation-exchange chromatography step. The recombinant inhibitor protein was shown to inhibit bovine mitochondrial F1-ATPase in a pH-dependent manner, as well as Saccharomyces cerevisiae mitochondrial F1-ATPase. Thorough analysis of the amino acid sequence revealed a potential coiled-coil structure for the C-terminal portion of the protein. Experimental evidence obtained by circular dichroism analyses supports this prediction and suggests F1I to be a highly stable, mainly alpha-helical protein which displays C-terminal alpha-helical coiled-coil intermolecular interaction.
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610.
G
G
Van Heeke
L
L
Deforce
R A
RA
Schnizer
R
R
Shaw
J M
JM
Couton
G
G
Shaw
P S
PS
Song
S M
SM
Schuster
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli; D005813:Genes, Synthetic; D008928:Mitochondria / Q000201:enzymology; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases