Cloning, overexpression, and characterization of cobrotoxin.
Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG-CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG-CBTX) was observed to undergo auto-catalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled (15N and 13C) and all the resonances ('H, 13C, and 15N) in the protein have been unambiguously assigned. ' H '5N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1H-15Nchemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the alpha7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders.
Hsieh
Hui-Chu
HC
Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan, ROC.
Kumar
Thallampuranam Krishnaswamy S
TK
Yu
Chin
C
D003001:Cloning, Molecular / Q000379:methods*; D003039:Cobra Neurotoxin Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D008970:Molecular Weight; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014170:Transformation, Genetic
D003039:Cobra Neurotoxin Proteins; D011993:Recombinant Fusion Proteins