Expression Study Of Porcine Peptidoglycan Recognition Protein-1 In Pichia Pastoris Based On Promoter And Chaperones

The methylotrophic yeast Pichia pastoris has been widely used for heterologous protein production.One of the main advantages of using P.pastoris as a protein production host is growing with methanol as its sole carbon and high cell-density.The most widely used promoter was alcohol oxidase?(AOX1)promoter which is remarkably strong and tightly regulated by methanol.Despite its favorable properties and industrial importance,transcriptional regulatory mechanism of AOX1promoter is not fully understood and needed further study.However,fine-tuning of heterologous gene expression is often needed to maximize protein expression level and building an efficient engineering for optimizing promoter activity should be based on a better understanding of regulatory mechanism of promoter.So it is difficult to obtain promoters with higher activities by traditional deletion/insertion strategy.Recent studies showed that poly(dA:dT)tracts could regulate yeast promoter activity by influencing the nucleosome occupancy and affinity,which affect the accessibility to transcription factor-binding sites.AOX1 promoter contain several poly(dA:dT)tracts.Part of this study was creating variants of AOX1 promoter by manipulating poly(dA:dT)tracts.Mammalian peptidoglycan recognition proteins(PGLYRPs)are highly conserved pattern-recognition molecules of the innate immune system with considerable bactericidal activity,which manifest their potential values for the application to food and pharmaceutical industry.In this study,Pichia pastoris X33was used to express porcine PGLYRP-1 and the activity of recombinant PGLYRP-1was also identified.The results of this study were presented as follows:1.Variants of AOX1 promoter were constructed by altering the presence and length of native poly(dA:dT)elements in different sites.To measure the promoter activity of these variants,we fused the promoters to reporter gene porcine growth hormone(pGH)and Lac Z respectively and the strength of promoter variants were measured by westernblot(pGH)and?-galactosidase activity(Lac Z).The results showed that deletion or lengthening native poly(dA:dT)tracts altered the variants activities ranging from~0.25 to~3.5 fold of wild-type promoter activity;If the activity of variant with deletion in one poly(dA:dT)site was less than or equal to wild-type promoter,addition of 15 bp poly(dA:dT)tract in the corresponding site could increase the promoter activity;If deletion in one poly(dA:dT)site could increase the promoter activity comparing with wild-type promoter,addition of 15 bp poly(dA:dT)tract in the corresponding site could reduce it to wild-type level or lower.In addition,deletion/addition of poly(dA:dT)tracts could affect nucleosome positioning and affinity.Deletion of poly(dA:dT)tracts increased affinity of nucleosome nearby the poly(dA:dT)tract,after addition of 15bp poly(dA:dT)tracts in different sites,there was a reduction in nucleosome occupancy upon addition of a nearby tract.Given that the transcriptional activity could be affected by the above changes to poly(dA:dT)tracts,we next asked whether these effects could be explained by predicted nucleosome architecture via manipulating poly(dA:dT)tracts.The cumulative sum of predicted nucleosome affinity across the region(-820 to-540)was proportional to promoters strength,while nucleosome affinity across the region((-620 to-430)was inversely proportional to promoters strength and low nucleosome affinity in region(-485 to-255)did not favor promoter activity.Overall,AOX1 promoter strength could be controlled by manipulating poly(dA:dT)tracts and deletion/addition of poly(dA:dT)tracts is an effective strategy to create promoter variants.2.To improve the production of recombinant PGLYRP-1 in P.pastoris,the coding sequence of PGLYRP-1 mature peptide was optimized according to codon usage bias of P.pastoris.Firstly,we explored the effect of gene dosage to the yield of PGLYRP-1,the results showed that increasing gene copy number of pglyrp-1decreased the amount of secreted protein in strains harboring multiple copies of pglyrp-1 gene and found that large amount of insoluble PGLYRP-1 protein accumulated inside the cell.In order to improve the secretion of PGLYRP-1,we investigated the effect of PDI and BiP on PGLYRP-1 secretion in different pglyrp-1copy strains,the results showed that overexpression of PDI could improve the secretion level of PGLYRP-1 protein and decrease insoluble intracellular PGLYRP-1level especially in high copy pglyrp-1 strains,but BiP could not improve the secretion level of PGLYRP-1 protein.In order to manifest the cause of the insoluble protein reduction,the transcript level of each gene was detected;the results showed that excessive expression of PDI and/or BiP could decrease the mRNA level of pglyrp-1gene.Taken together,on the one hand,PDI improved the folding of PGLYRP-1peptide.On the other hand,it could downregulate the transcription level of PGLYRP-1.Although BiP could not improve the yield of PGLYRP-1 protein,it also could downregulate the transcription level of PGLYRP-1.3.Finally,we performed 25 L bioreactor to investigate the expression of PGLYRP-1 in high cell density,purified PGLYRP-1 from culture medium and identified its biological activity.The results showed that the yield of PGLYRP-1 was not proportional to biomass and protein aggregation was observed when a certain level of PGLYRP-1 was reached.The yield of PGLYRP-1 was only 25 mg/L.Recombinant PGLYRP-1 could bind to some bacteria(both G~+and G~-)and inhibited the growth of certain bacteria.Our study laid the foundation for large-scale production and research on the activity of recombinant porcine PGLYRP-1.