Overexpression, purification, and pharmacological activity of a biosynthetically derived conopeptide.
A high yielding fusion protein system based on the protein cytochrome b(5) has been used for the production of novel 13-residue acyclic conopeptide. This peptide, Mo1659, can be liberated from the carrier protein using CNBr cleavage and subsequent purification using RP-HPLC methods. The yield of isotopically enriched peptides is high, ranging from 3 to 4mg of purified peptide from a 500ml culture, indicating that this system can be widely used for peptide production. Biosynthetic Mo1659 is active on non-inactivating K(+) channel much like the natural Mo1659, despite the absence of C-terminal amidation. Heteronuclear NMR studies show that the peptide exists in a conformational equilibrium involving proline-10. To our knowledge this is the first report of the production of an isotopically (15)N/(13)C-enriched conopeptide.
Kumar
Ganesan Senthil
GS
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Ramasamy
Palanisamy
P
Sikdar
Sujit K
SK
Sarma
Siddhartha P
SP
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D020916:Conotoxins / Q000737:chemistry*; D003488:Cyanogen Bromide / Q000737:chemistry; D015786:Cytochromes b5 / Q000737:chemistry; D017931:DNA Primers; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020916:Conotoxins; D017931:DNA Primers; D010455:Peptides; D015786:Cytochromes b5; D003488:Cyanogen Bromide