RESUMO
Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG-CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG-CBTX) was observed to undergo auto-catalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled (15N and 13C) and all the resonances ('H, 13C, and 15N) in the protein have been unambiguously assigned. ' H '5N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1H-15Nchemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the alpha7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders.
Assuntos
Clonagem Molecular/métodos , Proteínas Neurotóxicas de Elapídeos/biossíntese , Proteínas Neurotóxicas de Elapídeos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Neurotóxicas de Elapídeos/genética , Proteínas Neurotóxicas de Elapídeos/isolamento & purificação , Peso Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Transformação GenéticaRESUMO
In this report, we describe a simple approach to produce a large quantity of a recombinant cobra neurotoxin containing four pairs of disulfide bonds. A cDNA encoding the toxin was fused, in frame, to the carboxyl termini of thioredoxin via a linker sequence encoding two amino acids, Asp and Pro. Due to the presence of thioredoxin, a soluble form of the fusion protein was expressed in a compartment, sensitive to osmotic pressure, in Escherichia coli. The fusion protein was released into the solution with low ionic strength under an osmotic shock treatment, and purified in a single step using an ion exchange chromatography column. The purified protein was treated in diluted hydrochloric acid to induce hydrolysis of the protein at the Asp-Pro linker site. Then, the recombinant neurotoxin was purified by gel filtration of the acid-treated sample. When the biological activity of the purified toxin was assayed, it was as potent as the natural toxin. Using this protocol, approximately 12 mg of pure recombinant neurotoxin can be produced from one liter of bacterial culture. More importantly, this protocol can be easily used for the production of the toxin at a larger scale with low cost. The approach outlined in this report will be suitable for the production of other recombinant proteins especially those of the 'three-finger' family.
Assuntos
Proteínas Neurotóxicas de Elapídeos/genética , Escherichia coli/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Neurotóxicas de Elapídeos/biossíntese , Proteínas Neurotóxicas de Elapídeos/toxicidade , Dissulfetos/química , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Concentração Osmolar , Pressão Osmótica , Proteínas Recombinantes de Fusão/biossíntese , Tiorredoxinas/genéticaRESUMO
The novel cDNAs encoding four long neurotoxin homologues were firstly cloned from the venom gland of Naja naja atra by reverse transcriptase-polymerase chain reaction. Amino acid sequence comparison showed that they may come from two different evolutionary origins. The mature proteins were expressed as soluble fusion proteins in Escherichia coli and purified for immunoblotting. The results revealed that they displayed different immunochemical properties.
Assuntos
Proteínas Neurotóxicas de Elapídeos/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Proteínas Neurotóxicas de Elapídeos/biossíntese , DNA Complementar/química , Escherichia coli/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Neurotoxin-1 from Naja naja oxiana venom (NTX-1) has been crystallized by vapor diffusion in sitting drops. The crystals have cell dimensions of a = 25.2 A, b = 75.6 A, c = 35.9 A, and are in space group P2(1)2(1)2(1). Three-dimensional data to 1.9 A have been recorded by a Syntex P2(1) automatic diffractometer. The atomic structure of the toxin has been determined by molecular replacement using the alpha-cobratoxin (alpha-CTX) as the search model. The position of 534 non-hydrogen protein atoms have been determined. The model contains 65 water molecules. Refinement has led to an R-factor of 19.3% at 1.9 A resolution. The secondary and tertiary structures of NTX-1 have been analyzed and a comparison with structure of the alpha-CTX has been made.
