Expression And Optimization Of Kallikrein From Agkistrodon Halys Pallas Snake Venom Using A Wheat Germ Cell-free Protein Synthesis System

The genome DNA of kallikrein from Agkistrodon halys pallas snake venom hasbeen successful cloned. In this study, two restriction enzyme sites BamHI and XhoIand the His tag including six histidines have been added to the genome DNA ofvenom kallikrein. And the whole DNA was ligated into pCS²⁺, generating pCS²⁺/VK.The insertion was indentified by restriction analysis and sequencing.After the linearization of pCS²⁺/VK, the plasmid was transcribed in vitro and themRNA of venom kallikrein was obtained. Then the mRNA of venom kallikrein wasmixed with wheat germ extract and translation buffer and incubated at26°C for16h.After analyzed by SDS-PAGE electrophoresis and Western blot, the size ofrecombination venom kallikrein was about43kDa.Wheat germ cell-free protein synthesis system has the potential to synthesizefunctional proteins safely and with high accuracy, but the poor energy supply and theinstability of mRNA templates reduce the productivity of this system, which restrictsits applications. In this study, phosphocreatine and pyruvate were added to the systemto supply ATP as a secondary energy source. After comparing the protein yield, wefound that phosphocreatine is more suitable for using the wheat germ cell-free proteinsynthesis system. To stabilize the mRNA template, the plasmid vector, SP6RNApolymerase, and Cu2+were optimized, and a wheat germ cell-free protein synthesissystem with high yield and speed was established. When plasmid vector （30ng/μl）,SP6RNA polymerase （15U）, phosphocreatine （25mM）, and Cu2+（5mM） wereadded to the system and incubated at26°C for16h, the yield of venom kallikreinincreased from0.13mg/mL to0.74mg/mL.Recombination venom kallikrein was purified by Immobilized Metal AffinityChromatography （IMAC）. After dialysis and freeze-drying, the specific activity of therecombinant protein was1.3U/mg, which is only slightly lower than the crude venomkallikrein （1.736U/mg） due to the lack of the sugar chain.In this study, the yield of venom kallikrein was improved by optimizing thesystem, and a good foundation has been laid for industrial applications and for furtherstudies.