The Prokaryotic Expression Of Jingzhaotoxin-Ⅲ

The bioactive peptides isolated from venomous plants or animals areconsidered valuable drug candidates due to their high selectivity. In recent years,the study of spider peptic toxins demonstrated that they can be widely used inbasic research fields such as pharmacology, neuroscience and pharmaceuticalindustry. Currently, the spider toxins used for these purposes were eitherisolated from natural obtained venoms or chemically synthesized. Due to thelimited supply of natural spider venoms, by isolation from the venoms can hardlysatisfy the increasing needs of toxic components from both research andindustrial sides. Neither was the chemical synthesize strategy be able to solvethe problem not only because of its limited scale but also the highly expensivereagents required. With the development of modern molecular biology, thecloning and expression of peptic toxin were routinely performed in researchworks. Several expression systems were established over the past decades,including the E. coli, yeast, insect cell, mammalian cell, and plant cell systems.Chilobrachys Jingzhao is newly identified spider species found in China,and it mainly distributed in Guangxi and Hainan province. Its venom is acomplex mixture which includes many bioactive components. Jing Zhao toxin-Ⅲ(JZTX-Ⅲ), which is separated from crude venom, has36amino acid residuesand three pairs of cysteine. The complexity of its structure makes it hard to beexpressed in other cells using genetic engineering methods. Our team hassuccessfully expressed JZTX-Ⅲ toxin by using pET expression system. First,primers based on coding sequences of JZTX-Ⅲ toxin are designed. Then, JZTX-Ⅲ fragment was amplified by PCR method and then ligated to expression vector,the construct was thus named as pET40b-FrJZTX-Ⅲ. At last, the vector isintroduced to expression E.coli strain BL21(DE3). Two different inductionmethods have been used to express our target protein: IPTG induction andautomatic induction. By optimizing the condition of expression system, we findautomatically induction has obvious advantages in expressing toxin polypeptides:heterologous protein expression level has been greatly enhanced, and so has theproportion of soluble active ingredients. In addition, fusion proteins with DsbCtag is secreted into the E.coli periplasmic space, which can be separated by lowpermeability way. In this way, the purity is improved at the crude separation level. Nickel column affinity chromatography is applied for the primarypurification of fusion protein which is subsequently digested by enterokinase toget a complete intact toxin polypeptides and label. Later, a purified recombinantprotein rJZTX-Ⅲ is obtained through RP-HPLC. Finally, SDS-PAGEelectrophoresis and mass spectrometry demonstrate that our recombinant proteinhas the same molecular weight as its natural form. Further analysis of itsphysiological properties by using patch clamp also substantiates this consistency.In addition, after a series of research of inducing temperature, time and theshaking speed, we found that at a low temperature of25℃, the yield of solubleprotein can be greatly improved. The optimal time is20hours because E.colipopulation can get a high concentration. Also, rotation speed has little effect onthe expression.