Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus.
BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome b(5) fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage by Escherichia coli. High expression levels of the fusion protein enabled facile purification of this peptide. The presence of disulfide bonded isomers, occurring as distinctly populated states even in the fusion protein, were separated by gel filtration chromatography. The target peptide was liberated from the host protein by Tev protease cleavage and subsequent purification was achieved using RP-HPLC methods. Reverse phase HPLC clearly showed the presence of at least two isomeric forms of the peptide that were significantly populated. The oxidative folding of BTK-2 was achieved under ambient conditions during the course of purification. Structural characterization of the two forms, by solution homonuclear and heteronuclear NMR methods, has shown that these two forms exhibit significantly different structural properties, and represent the natively folded and a "misfolded" form of the peptide. The formation of properly folded BTK-2 as a major fraction without the use of in vitro oxidative refolding methods clearly indicate the versatility of the Cytochrome b(5) fusion protein system for the efficient production of peptides for high resolution NMR studies.
Kumar
G Senthil
GS
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Sarma
Siddhartha P
SP
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods; D004220:Disulfides / Q000302:isolation & purification; D010450:Endopeptidases / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017510:Protein Folding; D012604:Scorpion Venoms / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D012605:Scorpions
C549471:BTK-2 toxin, Mesobuthus tamulus; D004220:Disulfides; D010455:Peptides; D012604:Scorpion Venoms; D010450:Endopeptidases; C099488:TEV protease