Soluble Expression Of Serratia Marcescens Phospholipase A1 Accessory Protein And Interaction With Phospholipase A1

The phospholipase A1 accessory protein PlaS is a segment of the downstream sequence of the coding gene plaA,which is closely related to the high activity expression of phospholipase A1 in E.coli.In order to study the nature of accessory protein PlaS and its regulation mechanism for phospholipase A1 enzyme activity,SP28 recombinant plasmid was constructed.Experiments showed that the expression of the accessory protein PlaS in the recombinant plasmid could not be detected by SDS-PAGE,and only a very slight expression was detected by the Western Blot technique.In this dissertation,bioinformatics analysis was performed on the protein structure of the accessory protein PlaS,and it was determined that PCR was used to truncate the N-terminal 35 amino acids of the accessory protein PlaS and successfully construct the recombinant plasmid dSP28.The induced expression of the constructed recombinant plasmid dSP28,combined with optimization of the fermentation medium,and a large amount of soluble target protein was obtained.Finally,through the yeast two-hybrid technique,the construction of co-expressing strain d BP28,and Biacore protein interaction experiments,the in vitro and in vivo interaction properties of the accessory protein PlaS and phospholipase A1 were studied.The main research contents and results are as follows:?1?Construction of plasmin A1 accessory protein PlaS expression systemUsing bioinformatics methods,the plaS gene sequence was analyzed.In order to promote the intracellular soluble expression of phospholipase A1 accessory protein,the N-terminus of the accessory protein PlaS was truncated by 35 AA.The truncated dplaS gene was inserted into the vector pET-28a?+?,and the correct recombinant plasmids that were verified by sequencing were transformed into BL21?DE3?host bacteria for expression,and the recombinant expression strain dSP28 was successfully constructed.?2?Optimization of fermentation medium for phospholipase A1 accessory protein and protein purificationThe successfully constructed dSP28 strain was fermented and cultured.In order to make the target protein express in a large amount in the cell,the different components of the fermentation medium were compared and the optimal fermentation medium was TB plus 3%glycerol.The PlaS protein was induced to express in the fermentation medium,and the target protein was purified by AKTA system.The purified target protein concentration was 33.78?g/mL.?3?Study on the interaction between PlaA protein and accessory protein PlaS and dPlaS in vivoYeast two-hybrid technology was used to successfully construct bait plasmidpGBKT7-PlaA,preyplasmidsp GADT7-PlaSand pGADT7-d PlaS by molecular cloning.First,the bait plasmid pGBKT7-PLA was transformed into yeast competent cells for self-activation verification.If auto-activation reaction did not occur,then the bait plasmid pGBKT7-PlaA and the prey plasmid pGADT7-PlaS,pGADT7-d PlaS were co-transformed into Y2 HGold yeast competent Cells,and add negative and positive controls.The results showed that the bait protein did not self-activate.In the subsequent co-transformation experiment,the color of the colonies in the positive control group was the same as the colony color on the co-transformation plates of the bait plasmid and the prey plasmid,both of which were blue,while those in the negative control group.The colony color is white,indicating that PlaA protein interacts with PlaS protein and PlaA protein and dPlaS protein in vivo and activates the reporter gene in yeast,so there is an interaction between them.In order to further verify the interaction between PlaA and the accessory proteins PlaS and dPlaS in vivo,the co-expressing strain dSP28 of plaA gene and dplaS gene was constructed,and compared with the enzyme activity of phospholipase A1 constructed by the previous laboratory and containing the plaA gene and plaS gene overlapping strain BP28.It was found that the co-expressing strain d PlaS had a promoting effect on the expression of PlaA protein,and the activity of the enzyme was significantly increased.This shows that there is an interaction between the truncated accessory protein dPlaS and the PlaA protein.?4?Interaction between PlaA protein and accessory protein dPlaS in vitroThe AP28 recombinant plasmids constructed in the previous laboratory were cultured and cultured to obtain a large number of inclusion bodies of PlaA protein.The obtained inclusion bodies were subjected to renaturation,and the purified PlaA protein was purified using an AKTA protein purification apparatus.Meanwhile,a large number of soluble accessory proteins expressed and purified,and the purity of the purified PlaA protein and the accessory protein dPlaS reached more than90%.Subsequently,the purified protein was used to interact with each other on the Biacore protein interactor.It was found that the PlaA protein and the accessory protein d PlaS could bind specifically and showed good concentration dependence.The association rate constant ka between the two proteins was 9223 Ms^-1.The dissociation rate constant kd was1.054E-5 s^-1and the kinetics affinity KD was 1.143E-9 M.