Site-Directed Mutagenesis Analyses, Codon Optimization Study Of Harpin_x Protein

Harpins are a group of glycine-rich,heat-stable and protease K-sensitive proteins that are able to elicit disease and insect resistance in plants,induce many plant-reaction phenotypes,and promote plant growth in yield and quality.Harpin can induce HR in non-host plants,and may be an important pathogenicity factor of bacterial plant pathogen.In this study,we identified a key functional region in harpins from Xanthomonas that suppressed protein aggregation and mediated its expression in E.coli.Our data suggested that the presence of two common features in harpins(Wei et al 1992),namely,high glycine content and lack of cysteine residues,were not sufficient for Xanthomonas to elicit hypersensitive response(HR) activity or heat stability.Additionally,bioinformatic analyses revealed that the secondary structure of a conserved N-terminal region consisting of 12 highly hydrophilic amino acids(QGISEKQLDQLL) was a-helical.Following site-directed mutagenesis deletion of this region,the three mutated harpin proteins,in cultures induced at 37℃,failed to elicit a HR in tobacco leaves.However,at 24℃,two mutated harpins retained the ability to elicit HR,albeit with lower expression levels than that noted with the wild-type.SDS-PAGE and Western blot data suggested the HpaG mutant protein was found almost entirely in the inclusion body.These data demonstrated that these conserved amino acid residues played a critical role in protein aggregation and inclusion body formation in harpins from Xanthomonas.And then,following site-directed mutagenesis were carried out,the three mutated harpin proteins,HpaG(L50P),harpin_Xoo(L51P) and harpin_Xooc(L53P) failed to elicit a HR in tobacco leaves even at 10 uM.These results suggested that the 50^th,51^th and 53^th leucine amino acids(AA) of HpaG,harpin_Xoo and harpin_Xooc played a critical role in inducing the HR.The products of 9 mutated harpin_X fragments were tested for functions in pathogen resistance in tobacco.Results from the assay showed:the mutant proteins failed to induce HR still induced pathogen defense.Interestingly,HpaG(L50P),harpin_Xoo(L51P) and harpin_Xooc(L53P) protein can induce resistance to tobacco mosaic virus in tobacco. Furthermore,the products of 3 mutated harpin_X induced resistance to consistently, treatment with caused expression of the genes that encode regulators and effectors involved in defense signal transduction pathways in tobacco.In contrast,empty vector pET(30a+) treatment with did not induce expression of these genes.Intriguingly,harpin_Xooc(L53P) protein failed to induce HR can also induce expression of signaling-regulatory genes.These results suggest that the functional domains for the HR and pathogen defense act consistently for the phenotypes and molecular response.Among the many reported causes preventing efficient heterologous protein production in E.coli are biased codon usage,in addition,rare codon gene expression can lead to translational errors as a result of ribosomal stalling at a position requiring incorporation of amino acids coupled to minor tRNAs,or even at sites requiring major tRNAs,but which are depleted because of overutilization of a particular amino acid produced with expression plasmids encoding wild-type genes.So we synthesized codon-optimized hrf2 gene.This gene were ligated pET(30a+) expression vector,and the constructs were expressed in BL21 (DE3).But surprisingly hrf2_syn gene expression levels was less than hrf2 gene in BL21(DE3).Which prevented high level expression of the hrf2_syn gene? The results of reverse transcription PCR and real-time PCR analysis of codon-optimized gene mRNA implied the importance of mRNA secondary structure formation,but not necessarily tRNA abundance for efficient heterologous protein production in E.coli.In addition,rare codon genes optimization is a considerable robust alternative choice.But it is important to prevent problems encountered by specific RNA secondary structure formation.Harpin_X were purified using the Bulk GST purification module and Thrombin cleavage capture kit.The yield was more than 6 mg purified harpin_X proteins per liter of culture and the concentration of purified harpin_X proteins was about 1.5～2.0 mg/ml.Purified harpin_X proteins samples were at least 90 to 95%pure on a Coomassie stained SDS-PAGE.Then desalted by Desalting Columns,Molecules greater than 5,000 MW emerge in the void volume,separating them from the smaller molecules.The Pre-Screen Assay Suite for screening of optimal initial protein concentration.And protein crystals can form under a variety of conditions;finding the right one is often the challenge.A widely use means of growing crystals is called the hanging-drop vapor-diffusion method.A drop of protein solution containing a low concentration of a precipitant(such as a salt) is suspended above a reservoir containing a higher concentration of the precipitant.The exchange of water vapor between drop and reservoir(vapor diffusion) results in shrinkage of the drop,which raises the protein concentration,and also raises the concentration of precipitant in the drop toward the concentration of precipitant in the reservoir.When protein concentration exceeds the solubility of the protein in the drop, protein precipitates.If the protein is pure and precipitation is slow,crystals of the protein may form and grow.Under less optimal conditions,the protein may precipitate as an amorphous solid or gel,which is useless for structure determination.Our goal is to optimize the growth conditions for harpin_X proteins.Utilized Crystal Screen Kit(Hampton Ltd.),we found some rudimental results:the solution containing PEG and AmSO₄ of a precipitant were easily induced harpin_X proteins precipitated.These suggested us to optimize the growth conditions from the precipitant solution containing PEG(2000,4000,8000) or AmSO₄.And on the other hand,we need test more precipitant so as to get crystals of harpin_X proteins.