Sodium Current Modulation Of Scorpion Polypeptide BmK IT And BmK IT-AP From Recombinant Protokaryon Expression

Scorpion toxin molecule is a polypeptide aggregate by the 28 to 76 amino acid residues, showing a variety of biological characteristics. α-like is generally considered a long-chain peptide scorpion toxins act on sodium channels, slow inactivation of sodium channels, from the pain caused by the effect, while the β-like of long-chain polypeptide scorpion toxin opposite. Sodium channels play an important role in neuronal excitability and other produce regulation and action potentials. Sodium channel disease is due to a gene encoding the sodium channel mutation or abnormal expression, or within the environment occurs when sodium channel pathogenic substance, structure or function of sodium channels in different degrees of abnormal, leading to the body’s physiological function disorder, caused by congenital or acquired disease. Thus, the functions and characteristics of sodium channel for people to overcome some of the sodium channel disease in search of better treatment, has a very important practical significance. One important way to study the natural scorpion toxin sodium ion channels, but because of natural scorpion toxin protein was more difficult, and thus the study of sodium ion channels have also been very limited. As a result, the scorpion toxin polypeptide gene recombinant expression and transformation is sodium channel structure and the function of one of the effective ways to study.By the scorpion, Buthus martensii Karsch(Bm K) cloned into two kinds of e xcitatory insect toxin Bm K IT and Bm K IT-AP, which Bm K IT toxin mature pep tide of 69 amino acid residues, of which four disulfide bridges, a major role in i nsect cell membrane sodium ion channels, causing repeated firing action potentials, enhance sodium peak current, peak conduction delay sodium closure, insect excit atory paralysis to death. Bm K IT-AP mature peptide toxin contains 72 amino acid residues and four disulfide bridges, belongs to a class of excitatory insect s elective toxin. In this study, the gene sequence with His6 at the end of two kind s of scorpion toxin polypeptide cloned into the vector p ET-32(a), the successful re combinant plasmid p ET-32(a)-N-Tag(His)-Bm K IT and p ET-32(a)-N-Tag(His)-Bm K IT-AP. Then, these two plasmids were transformed into E.coli BL21(DE3) in so luble prokaryotic expression, and expression induced by optimizing time, IPTG co ncentration, temperature induced optimal expression condition; and by buffer flow rate ratio get a lot of optimization and reorganization of toxins Bm K IT and Bm K IT-AP. By cell patch clamp detection depth study of their physiological and ch emical properties, mainly following results were obtained.According to Genbank scorpion toxin polypeptide Bm K IT and Bm K IT-AP mature peptide sequence designed and optimized codon preference in accordance with the synthetic gene sequence Bm K IT and E. coli codon Bm K IT-AP to cons truct the expression vector p ET-32(a)-N-Tag(His)-Bm K IT and p ET-32(a)-N-Tag(Hi s)-Bm K IT-AP.The recombinant Bm K IT theoretical molecular weight is 28553.333 Da. The recombinant protein Bm K IT-AP theoretical molecular weight 28113.3333 Da. De sign of experiments by inducing conditions, draw scorpion neurotoxin Bm K IT op timal expression conditions: induced time was 8 h, IPTG 0.5 mmol/L, temperature of 20℃, conditions induce protein expression of total protein the 43.5%; scorpio n neurotoxin Bm K IT-AP expression conditions: induced time 5 h, IPTG 0.5 mm ol/L, temperature-induced protein expression purposes under the conditions of 25℃the total amount of protein 44.1%.Metal ions Ni2+ affinity chromatography initial separation from the cell super natant after centrifugation of purified recombinant scorpion toxin polypeptide Tag(His)-Bmk IT and Tag(His)-Bmk IT-AP; sample pretest restructuring scorpion toxin polypeptide Tag(His)-Bmk IT cell supernatant at a concentration of 30 μg/ml, t he cells supernatant 10 ml of through metal ions Ni2+ affinity chromatography, elut ed peaks freeze-drying process to obtain 10 μg recombinant scorpion toxin polype ptide Tag(His)-Bmk IT, the yield is about 33.3%; the sample pretest recombinant scorpion toxin polypeptide Tag(His)-Bmk IT-AP cell supernatant at a concentration of 35 μg/ml, 10 ml of bacterial cells on supernatant through metal ions Ni2+ affi nity chromatography, eluted peaks freeze-drying process to obtain 12 μg recombin ant scorpion toxin polypeptide Tag(His)-Bmk IT-AP, the yield is about 34.3%; Th en the Ni2+ affinity chromatography eluting peaks RT-HPLC to give pure fractions electrophoresis recombinant scorpion toxin polypeptide Tag(His)-Bmk IT and Tag(His)-Bmk IT-AP.Cell patch clamp assay showed that the 10 μmol/L of recombinant scorpion toxin polypeptide Tag(His)- Bm K IT role in rat DRG, compared with the control group, the activation time is slightly delayed, peak sodium current value slightly increased, and 10 μmol/L of recombinant scorpion toxin polypeptide Tag(His)- Bm K IT-AP effects on rat DRG, treatment group was significantly higher than the control group sodium peak current values decreases while the activation time was prolonged, the mechanized, Tag(His)- Bm K IT cell of SD rat DRG sodium current suppression no significant adjustment, Tag(His)- Bm K IT-AP cell membrane of SD rat DRG sodium current has modulating inhibitory activity.