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Bacterial Production and Refolding from Inclusion Bodies of a “Weak” Toxin, a Disulfide Rich Protein

E. N. Lyukmanova1*, M. A. Shulepko1,2, R. V. Tikhonov1, Z. O. Shenkarev1, A. S. Paramonov1, A. N. Wulfson1, I. E. Kasheverov1, T. L. Ustich1,3, Yu. N. Utkin1, A. S. Arseniev1, V. I. Tsetlin1, D. A. Dolgikh1*, and M. P. Kirpichnikov1,2

1Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 330-6983; E-mail: katya@nmr.ru; dolgikh@nmr.ru

2Lomonosov Moscow State University, 119991 Moscow, Russia

3Medical Center of Denver University, USA

* To whom correspondence should be addressed.

Received September 26, 2008; Revision received November 14, 2008
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 125I-labeled α-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (α12β1γδ) showed the presence of biological activity of the recombinant “weak” toxin close to the activity of the natural toxin (IC50 = 4.3 ± 0.3 and 3.0 ± 0.5 µM, respectively). The interaction of the recombinant toxin with α7 type human neuronal acetylcholine receptor transfected in the GH4C1 cell line also showed the presence of activity close to that of the natural toxin (IC50 31 ± 5.0 and 14.8 ± 1.3 µM, respectively). The developed bacterial system for production of N. kaouthia venom “weak” toxin was used to obtain 15N-labeled analog of the neurotoxin.
KEY WORDS: neurotoxins, nicotine acetylcholine receptor, bacterial expression, refolding

DOI: 10.1134/S0006297909100101