The Expression, Purification And Primer Structure Analysis Of Protein Eliciter PebC1

Protein elicitor is a type of protein derived from pathogenic fungi, which can interact with plant and induce plant defense response, accelerate plant metabolism and promote plant growth. PebC1 is a novel protein elicitor derived from Botrvtis cinerea by our laboratory. In this study, we did cloning and expression of PebC1 and bcβNAC which have an interaction with PebC1 with prokaryotic and eukaryotic expression system. Then the coexpression of the two proteins was made. After purification, primary structure analysis of PebC1 and bcβNAC protein was done by a serials assay. The main results are as follows.The amino acid sequence of PebC1 was analysed by bioinformatic tools and cloned the gene egd1 witch encoded the bcβNAC based on the online database.The prokaryotic expression system is suitable for PebC1 and bcβNAC protein expression. Amplified gene pebC1 and egd1from Botrvtis cinerea were ligated to expression vector pET-30His and pET-30His+MBP respectively. The recombinant plasmids were transformed into expression strain Rosetta. The two genes were also inserted into pFastBac donor vectors, and prepared recombinant baculovirus stocks respectively. Then the recombinant baculovirus were used to transfect High Five(Hi5)cells for expression.The prokaryotic expression system is suitable for PebC1 and bcβNAC complex protein expression. The gene egd1and pebC1 were inserted into the mult-cloning site 1 and mult-cloning site 2 of pETDuet-1 vector respectively. Then the recombinant plasmid was transformed into expression strain Rosetta for coexpression. The two recombinant baculovirus also used to transfected Hi5 cells simultaneously for coexpression. But no complex protein can get due to the rapid digestion of PebC1 in insect cells. The purifications of all those proteins were realized. Those proteins were purified by affinity chromatography. Then HiTrap Q HP column was used for anion exchange chromatography. Finally size exclusion chromatography was performed through Superdex 200 column. The highly purified proteins were prepared for next experiments.The dot matrix structure of PebC1 and mbp-bcβNAC was determined by small angle scattering analysis. According to the homology model of NAC domain and UBA domain, we assigned the homodimer of NAC domain formed into aβbarrel in the central of the protein. Other parts except the UBA domain tightly surround the barrel. The UBA domains were hanged by the side. According to the dot matrix model of mbp-bcβNAC, the structure was divided into two parts: Part 2 forms into a cylindrical with 50A diameter; Part 1 forms into platykurtic with 30? depth which match the structure of MBP protein. So MBP protein was assigned into Part 1. But the exactly structure of bcβNAC remain unclear.Twinned crystals of complex protein were obtained. Purified heterodimer protein of PebC1 and bcβNAC was set up to grow crystals by sitting-drop vapor-diffusion method at 20℃. Twinned crystals were emerged in two conditions, which were Crystal Screen II 19# and MolecularDimensions MD1-02 34#. The results facilitate the structure research of PebC1 and its complex protein with bcβNAC.