Expression,Purification And Crystallization Of Intracellular And Extracellular Ends Of PiGK5 Protein From Phytophthora Infestans

Potato late blight has surpassed wheat rust and rice blast and has become the first crop disease in the world.The Pi GK5 protein of P.infestans belongs to the GPCR-PIPKs(GKs)protein family.It is shown that the protein plays an important role in sexual reproduction,asexual reproduction and pathogenicity of P.infestans.However,the study on the structure of Pi GK5 protein of Phytophthora infestans has not been reported so far.It is well known that transmembrane proteins are usually expressed in the form of inclusion bodies with no biological activity when expressed in prokaryotic system.Therefore,in this study,extraxellular and intracellular end of Pi GK5 protein and three deleted mutants of its intracellular end(intracellular-H8-LRx GI-PIPK protein,intracellular-LRx GI-PIPK protein and intracellular-PIPK protein)were expressed in prokaryotic system,purified and crystallized.Firstly,the c DNA coding regions of the extracellular,intact intracellular and intracellular ends of Pi GK5 protein were amplified by PCR,and the amplified fragments were cloned into the prokaryotic expression vectors p ET30 and p TEV,respectively.Then,the prokaryotic expression vectors of each protein were transformed into E.coli respectively and induced to express.After that,the protein was extracted and purified by nickel column and protein purification system.Finally,the purified proteins were screened by crystal screening.The results showed that the extracellular-His and the deletion mutant PIPK-His of intracellular end of Pi GK5-mainly expressed in the inclusion bodies,and only a small amount existed in the form of soluble protein in the supernatant.The Pi GK5-intracellular end-His and its deletion mutants H8-LRx GI-PIPK-His and LRx GI-PIPK-His exist only in the form of inclusion bodies in the precipitation.Pi GK5-extracellular end-MBP-His protein was only expressed as soluble protein in E.coli,and it is expression level was high,but the protein was degraded slightly.In E.coli,Pi GK5-intracellular end-MBP-His protein and its deletion mutants LRx GI-MBP-His protein and PIPK-MBP-His protein mainly existed in the form of inclusion bodies in the precipitation,only a small amount of soluble protein in the supernatant,and Pi GK5-intracellular end-MBP-His protein was almost completely degraded.However,the intracellular terminal deletion mutant H8-LRx GI-PIPK-MBP-His protein exists only in the form of inclusion body in E.coli.The results demonstrated that MBP tag had obvious solubilization effect on the extracellular end of Pi GK5 protein.The addition of 5% surfactant during cell fragmentation and 1%surfactant during protein elution can effectively promote the solubility of extracellular-His and intracellular-PIPK-His protein of Pi GK5.At the concentration of 12.0mg/m L,the extracellular-MBP-His protein can form a small number of regular rhombohedral crystals,while at the concentration of 8.5mg/m L,the intracellular-PIPK-His protein can form more regular rhombohedral crystals.The completion of this work lays a foundation for the study of the structure of Pi GK5 protein of Phytophthora infestans,and is of great significance for further revealing the function of the protein and further developing the targeting drugs of Pi GK5 protein for effective disease control.