Cloning, direct expression, and purification of a snake venom cardiotoxin in Escherichia coli.
The cardiotoxin analogue III (CTX III), isolated from the Taiwan cobra (Naja naja atra) venom, is a sixty-amino acid, all beta-sheet protein. We report the direct expression of CTX III from its synthetic gene as inclusion bodies in Escherichia coli. The yield of the expressed protein is about 40 mg/liter of the culture. CTX III trapped as inclusion bodies is dissolved and refolded by the slow refolding technique. The refolded protein is purified by reverse phase high performance liquid chromatography. The purified and refolded CTX III sample is further characterized by SDS-PAGE, circular dichroism, two-dimensional NMR spectroscopy and haemolytic activity. To our knowledge, this is the first report of the direct expression and purification of snake venom cardiotoxins.
Kumar
T K
TK
Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan.
Yang
P W
PW
Lin
S H
SH
Wu
C Y
CY
Lei
B
B
Lo
S J
SJ
Tu
S C
SC
Yu
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004179:Cobra Cardiotoxin Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D017931:DNA Primers; D017815:Elapidae; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006461:Hemolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D004179:Cobra Cardiotoxin Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; C082202:cardiotoxin III, Naja naja atra