Bacterial production and refolding from inclusion bodies of a “Weak” toxin, a disulfide rich protein

Abstract

The gene for the "weak" toxin of Naja kaouthia venom was expressed in Escherichia coli. "Weak" toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of "weak" toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia "weak" toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain "weak" toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of (125)I-labeled alpha-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (alpha1(2)beta1gammadelta) showed the presence of biological activity of the recombinant "weak" toxin close to the activity of the natural toxin (IC(50) = 4.3 +/- 0.3 and 3.0 +/- 0.5 microM, respectively). The interaction of the recombinant toxin with alpha7 type human neuronal acetylcholine receptor transfected in the GH(4)C(1) cell line also showed the presence of activity close to that of the natural toxin (IC(50) 31 +/- 5.0 and 14.8 +/- 1.3 microM, respectively). The developed bacterial system for production of N. kaouthia venom "weak" toxin was used to obtain (15)N-labeled analog of the neurotoxin.

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

“…Cloning and Bacterial Expression of WTX Mutants-The mutations were inserted into the WTX gene by site-directed mutagenesis using the expression vector pET22b(ϩ)/wtx (17) as a template. The cultivation of Escherichia coli BL21(DE3) cells transformed with appropriate vectors, gene expression, purification, and refolding of rWTX and its mutants were as described previously (17), with the exception that renaturation buffer contained additionally 0.5 M L-Arg.…”

“…The cultivation of Escherichia coli BL21(DE3) cells transformed with appropriate vectors, gene expression, purification, and refolding of rWTX and its mutants were as described previously (17), with the exception that renaturation buffer contained additionally 0.5 M L-Arg. For production of the 15 N-labeled rWTX and P33A mutant, transformed cells were grown on TB medium until A 600 0.6.…”

“…It has been shown on the examples of rWTX, water-soluble domain of human Lynx1 protein, and secreted Ly-6/uPAR related human proteins SLURP-1 and SLURP-2 that the expression in the form of E. coli inclusion bodies with subsequent refolding represents the optimal strategy for production of three-finger proteins containing an additional fifth disulfide bond in the loop I (17,(33)(34)(35). Therefore, to produce rWTX mutants we used a previously developed protocol (17), but to increase the refolding yield, the renaturation buffer was additionally supplemented with 0.5 M L-Arg.…”

“…Therefore, to produce rWTX mutants we used a previously developed protocol (17), but to increase the refolding yield, the renaturation buffer was additionally supplemented with 0.5 M L-Arg. The final yield of the refolded toxins was ϳ1-5 mg (for different mutants) per 1 liter of bacterial culture.…”

NMR Structure and Action on Nicotinic Acetylcholine Receptors of Water-soluble Domain of Human LYNX1

“…E. coli BL21(DE3) cells transformed with pET-22b(ϩ)/wslynx1 vector were grown at 37°C on Terrific Broth medium using a fermenter (Bioflow 3000, New Brunswick Scientific) under automatic maintenance of oxygen content in the system at a level of 30%. Gene expression was induced by addition of isopropyl 1-thio-␤-D-galactopyranoside to a final concentration of 0.025 mM at A 600 0.6, and cells were grown additionally for 18 h. Ws-LYNX1 was purified and refolded as was described for nonconventional neurotoxin WTX from Naja kaouthia venom (17). Briefly, ws-LYNX1 was extracted from inclusion bodies after incubation with 50 mM NaP i , 8 M urea, 1 mM tris(2-carboxyethyl)phosphine, 5 mM DTT, pH 7.4.…”