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, 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.
Drevet
P
P
Département d'Ingénierie et d'Etudes des Protéines, DSV, Saclay, CEA, Gif-sur-Yvette, France.
Lemaire
C
C
Gasparini
S
S
Zinn-Justin
S
S
Lajeunesse
E
E
Ducancel
F
F
Pinkasfeld
S
S
Courçon
M
M
Tremeau
O
O
Boulain
J C
JC
Ménez
A
A
D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D004546:Elapid Venoms / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004870:Erabutoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D009498:Neurotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009587:Nitrogen Isotopes; D011485:Protein Binding; D017510:Protein Folding; D011950:Receptors, Cholinergic / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics; D014170:Transformation, Genetic; D010738:Type C Phospholipases / Q000378:metabolism
D004546:Elapid Venoms; D009498:Neurotoxins; D009587:Nitrogen Isotopes; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C038908:erabutoxin A; D004870:Erabutoxins; D010738:Type C Phospholipases; D003488:Cyanogen Bromide