HIGH-LEVEL PRODUCTION AND ISOTOPE LABELING OF SNAKE NEUROTOXINS, DISULFIDE-RICH PROTEINS

The aim of this work was to produce and to label snake neurotoxins, di sulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was us ed as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-bi nding domain of protein A (B. Nilsson et QI., 1987, Protein Eng. 1, 10 7-113), thus preventing degradation in the bacterial cytoplasm and pro viding a simple affinity-purification method on IgG Sepharose. A solub le fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin. The toxin moiety was folded on the column while the hybrid was still bound. The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but With a need for red ucing molecules. The concentration of the hybrid bound to the column c ould be increased up to 3.3 mg/ml without significantly altering the f olding process. CNBr cleavage of the fusion protein followed by a puri fication step yielded about 2 mg of biologically active toxin mutant p er gram of dry cell weight. This procedure was applied to produce 55 m g of a toxin uniformly labeled with N-15. (C) 1997 Academic Press.