Cloning and expression of wild-type and mutant forms of the cardiotonic polypeptide anthopleurin B.
Venom of the sea anemone Anthopleura xanthogrammica contains a minimum of three polypeptide toxins capable of prolonging the repolarization phase of the action potential. A synthetic gene for the most toxic of the Anthopleura toxins, anthopleurin B (ApB), has been designed, synthesized, and expressed as a fusion protein with the gene 9 product of bacteriophage T7 in Escherichia coli. The fusion protein has been purified and its disulfide bonds reoxidized using glutathione redox couples. Upon cleavage with staphylococcal protease, this protocol yields approximately 1 mg of native ApB/liter of original culture. The recombinant protein has been shown to be identical to natural ApB with respect to amino acid composition, amino-terminal sequence, secondary structure, high pressure liquid chromatographic mobility, and biological activity. A second form of ApB containing additional residues of glycine and arginine at its amino terminus has also been characterized. This protein, termed GR-ApB, is identical in specific activity to the wild-type form. This work lays the groundwork for a detailed analysis of ApB structure and action by site-directed mutagenesis.
Gallagher
M J
MJ
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267.
Blumenthal
K M
KM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002316:Cardiotonic Agents / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000494:pharmacology; D012615:Sea Anemones / Q000235:genetics*; D012964:Sodium / Q000378:metabolism; D013604:T-Phages / Q000235:genetics; D014701:Veratridine / Q000494:pharmacology
D002316:Cardiotonic Agents; D036341:Intercellular Signaling Peptides and Proteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014701:Veratridine; C026132:anthopleurin B; D012964:Sodium