Structure Of Accessory Protein S And Its Effect On The Expression Characteristics Of Phospholipase A1

The accessory protein encoded by the plaS gene of Serratia marcescens can significantly improve the expression activity of phospholipase A1 in E.coli,but it can inhibit the growth of host bacteria,and the inhibition mechanism has not been reported.The key areas of its inhibition were determined by the analysis of phospholipase A1 auxiliary protein S(PlaS)bioinformatics and the growth characteristics of N-terminal truncated series of expression strains,and the inhibition mechanism was preliminarily investigated by scanning electron microscopy(SEM)and flow cytometry(FCM).At the same time,the effect of the truncated proteins on phospholipase A1 was studied by yeast two hybrid and Biacore.The activity of phospholipase A1 of co-expression strain dB27P28(phospholipase A1 and PlaS of N-terminal truncated 27 amino acids co-expression)was determined to study the effect of truncated helper protein on the activity of phospholipase A1.The main research contents and results are as follows:(1)After the analysis of PlaS signal peptide,transmembrane region and hydrophobic region,23,24,25,26 and 27 amino acids were truncated at the N-terminal of PlaS respectively,and the truncated auxillary protein genes were respectively introduced into E.coli BL21(DE3).Among the constructed expression strains,dS23P28?dS24P28?dS25P28?dS26P28 still showed growth inhibition in varying degrees,while dS27P28 did not show growth inhibition,which initially showed that the first 27 amino acids at the N-terminal of PlaS were the key regions of inhibition.(2)SP28 and dS27P28 were induced and expressed,and P28 was used as control,then observed by SEM.The results showed that PlaS expression had serious damage to the cell membrane of E.coli.However,PlaS of 27 amino acids truncated from N-terminal expression had no effect on E.coli cell membrane damage.At the same time,flow cytometry data also showed that the rate of cell membrane damage reached 66.92%when SP28 strain was induced to express for 8 hours.These results are consistent with those of growth characteristics.(3)The bait plasmids pGBKT7-PLA1 and prey plasmids pGADT7-dPlaS27 were constructed by yeast two hybrid technique and transferred into Y2H gold yeast cells.The co transformed cells can grow on the QDO plate of TDO/3'at 20mm,which indicates that the expression of his and ade2 is activated,so there is interaction between PLA1 protein and dPlaS27 protein in vivo.After purification of PLA1 protein and dPlaS27 protein,the pure protein was interacted with each other in the Biacore protein interaction instrument.The results showed that the protein plaal and dPlaS27 could specifically bind,and showed a good concentration dependence,indicating the interaction between PLA1 protein and dPlaS27 in vitro.(4)A co-expression strain dB27P28 was constructed and compared with the phospholipase A1 activity of co-expression strain BP28(phospholipase A1 and PlaS co-expression).It was found that the activity of phospholipase A1 was greatly increased,which indicated that the function of phospholipase A1 was not changed after the 27 amino acids of PlaS were truncated,and the activity of phospholipase A1 was improved.Then OD600 value of initial induced cells,induction time and IPTG concentration of the co-expression strain dB27P28 were optimized,The results showed that when OD600 was 0.6 and 0.15mmol/l IPTG was added,induced expression 8 hours the enzyme activity reached a high level.The results of this study provide a theoretical basis for the further study of the functions PlaS of Serratia marcescens.