Bacterial production and refolding from inclusion bodies of a "weak" toxin, a disulfide rich protein.
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.
Lyukmanova
E N
EN
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.
Shulepko
M A
MA
Tikhonov
R V
RV
Shenkarev
Z O
ZO
Paramonov
A S
AS
Wulfson
A N
AN
Kasheverov
I E
IE
Ustich
T L
TL
Utkin
Yu N
YN
Arseniev
A S
AS
Tsetlin
V I
VI
Dolgikh
D A
DA
Kirpichnikov
M P
MP
D000818:Animals; D001419:Bacteria / Q000235:genetics* / Q000378:metabolism; D002038:Bungarotoxins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D004546:Elapid Venoms / Q000235:genetics* / Q000378:metabolism; D017815:Elapidae / Q000097:blood; D005822:Genetic Vectors; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation
D002038:Bungarotoxins; D004220:Disulfides; D004546:Elapid Venoms; C521753:Naja kaouthia venom