Expression Of Analgesic-antitumoral Peptide BmK AGAP Neurotoxin From Scorpion Buthus Martensii Karsch In Pichia Pastoris

The Asian scorpion Buthus martensi Karsch（BmK）is widely distributed in China,the venom is rich sources of toxic polypeptides that affect ion-channel function of excitable cells, containing 20-80 amino acid residues cross-linked by four or three disulfide bridges.Four different families of toxins have been described,which specifically interact with ion channels: Na⁺,K⁺,Cl^-,Ca²⁺and are improved to be tools for clarifying the physical effects and functional mapping of ion channels.The toxin is also the perfect model for the research on the Protein Structure and Function.However,it is difficult to purify for the complexity of toxin component.Meanwhile the content of useful toxins is limited.We can solve these problems by the genetic engineering methods.The objection of this study is to build an efficient expression system on the use of Pichia pastoris and to lay a foundation of further study on the prepare of the active scorpion.The gene sequence encoding mature peptide of BmK AGAP was amplified by PCR and inserted into the pPIC9K expression vector.To consider the inefficiency of Ste13 protease when desired protein expressed in Pichia pastoris,we construct another comparative vector to avoid the additional tetrapeptide（Glu-Ala-Glu-Ala）at N-terminal of the secreted protein.Then the recombination vectors were transformed to Pichia pastoris GS115 strains.After several screenings of grads of G418,the transformed stains containing multiple inserts were chosen to be induced with methanol in shake flask culture.The supernatant by SDS-PAGE suggests AGAP gene possible expression in Picha pastorsi GS 115 and KM71.It is the main method that using E.coil expression system to gain recombined scorpion toxins today.We utilized the Pichia pastoris expression system to express the scorpion toxin.And it is proved the expression system is feasible in technology,which pioneered the wide prospect of the exploitation and utilization of the abundant scorpion medicaments.