High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins

Protein Expr Purif. 1997 Aug;10(3):293-300. doi: 10.1006/prep.1997.0740.

Abstract

The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B. Nilsson et al., 1987, Protein Eng. 1, 107-113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose. A soluble 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 reducing molecules. The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process. CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight. This procedure was applied to produce 55 mg of a toxin uniformly labeled with 15N.

MeSH terms

  • Binding, Competitive
  • Chromatography, Affinity
  • Circular Dichroism
  • Cyanogen Bromide
  • Elapid Venoms / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Erabutoxins / biosynthesis*
  • Erabutoxins / chemistry
  • Erabutoxins / genetics
  • Erabutoxins / metabolism
  • Escherichia coli / genetics
  • Gene Expression
  • Genetic Vectors
  • Isotope Labeling
  • Magnetic Resonance Spectroscopy
  • Neurotoxins / biosynthesis*
  • Neurotoxins / chemistry
  • Neurotoxins / genetics
  • Neurotoxins / metabolism
  • Nitrogen Isotopes
  • Protein Binding
  • Protein Folding
  • Receptors, Cholinergic / metabolism
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Staphylococcal Protein A / chemistry
  • Staphylococcal Protein A / genetics
  • Transformation, Genetic
  • Type C Phospholipases / metabolism

Substances

  • Elapid Venoms
  • Neurotoxins
  • Nitrogen Isotopes
  • Receptors, Cholinergic
  • Recombinant Fusion Proteins
  • Staphylococcal Protein A
  • erabutoxin A
  • Erabutoxins
  • Type C Phospholipases
  • Cyanogen Bromide