Pegylation Of Cobra Neurotoxin

Polyethylene glycol (PEG) modification technology has been successfully applied to improving the performance of several protein drugs. In order to solve the problems which Cobra Neurotoxin (CNT) encountered in clinical applications (such as slow action and unremarkable analgesic effect), this PEG modification technology was used on CNT.CNT was purified from Cobra coarse venom by a simple two-step chromatographic method, which was a combination of one cation exchange chromatography (Source 30S) and one gel filtration chromatography (Superdex 75). It showed one single band on SDS-PAGE gel, and 100% purity on RP-HPLC chromatogram. The total yield was 22.4%. higher than formerly reported 12%.Four activated mPEG reagents (mPEG-SPA, mPEG-SMB, mPEG-ButyrALD. and mPEG2-NHS) were selected to modify the amino groups in CNT molecule. The optimization of reaction conditions was carried out for the set goal — the highest proportion of single-PEGylated CNT. mPEG-SPA was finally confirmed as the best modifier for CNT. Optimized reaction conditions were as follows: pH8.0, 28℃, CNT/ mPEG (molar ratio)=1: 5 and reaction time=1h. Among the four influencing factors, CNT/mPEG was the most significant one. while pH was the weakest. The PEGylated product was separated by cation exchange chromatography (Source 30S).The molecular weight, stabilities, immunogencity, and efficiency of the PEGylated producl were tested. And its modified site was primarily studied. SDS-PAGE and MALDI-TOF-MS proved this product to be single-PEGlated. Compared to natural CNT, the PEGylated one showed higher stabilities to repeated freeze-thaw and heat, greatly lowered immunogencity and toxicity. and better analgesic effects under same administration dosages and at same time points. The characters above mentioned revealed the advantage of PEGylation.