Expression and mutagenesis studies of cobrotoxin from Taiwan cobra.
The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra (Taiwan cobra). The cDNA was subcloned into the expression vector pET20b(+) and transformed into BL21(DE3) Escherichia coli strain. Expressed cobrotoxin was isolated from inclusion bodies of E. coli and subjected to refolding into its folded structure. The refolded cobrotoxin was purified by high-performance liquid chromatography and exhibited a neurotoxicity in inhibiting acetylcholine-induced muscle contractions. Recombinant cobrotoxin showed a tendency to isomerize its disulfide bonds as that observed with native cobrotoxin. An appreciable decrease in the rate of isomerization reaction was observed when Glu-38 was replaced with Gln-38 or Lys-47 was replaced with Glu-47 or Gln-47. These results reflect that the element in controlling the disulfide isomerization of cobrotoxin is closely associated with the charged side chains in the cobrotoxin molecule.
Chang
L S
LS
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan, 804. lschang@mail.nsysu.edu.tw
Chen
K C
KC
Wu
B N
BN
Lin
S K
SK
Wu
P F
PF
Hong
Y R
YR
Yang
C C
CC
D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry* / Q000235:genetics* / Q000633:toxicity; D017815:Elapidae; D004926:Escherichia coli; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics; D011898:Ranidae; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000633:toxicity; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013624:Taiwan
D003039:Cobra Neurotoxin Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000109:Acetylcholine