Functional Analysis,Heterologous Expression And Purification Of Yeast Lipase Opl1p

The balance of lipid metabolism is very important for the normal growth of cells.Lipid metabolism mainly depends on the synthesis and degradation of lipids to maintain the dynamic balance,and there are two main ways of lipid degradation,namely lipolysis and lipophagy.The lipolysis process plays an important role.In this experiment,a potential yeast lipase Ydr444 wp was discovered and named Opl1p(Oleate-induced LD targeting and Pet10 p interacting lipase 1)according to its functional properties.Through the analysis of the primary protein sequence of Opl1 p,the existence of the lipase marker sequence GXSXG was searched and determined,and the protein secondary and tertiary structure of Opl1 p was predicted by homology modeling.After sequence alignment and phylogenetic tree construction,A monoacylglycerol lipase Rog1 p with high affinity with Opl1 p was found,with a homology of 24.49 %.It is speculated that Opl1 p may be a monoacylglycerol lipase.In order to determine the localization and expression level of Opl1 p in Saccharomyces cerevisiae cells,a fluorescent-labeled Opl1 p stably expressed Saccharomyces cerevisiae strain Opl1-ym Cherry was constructed,and the yeast Opl1-ym Cherry was cultured in oleic acid medium.Droplets co-localize.Further,Opl1-ym Cherry was cultured in oleic acid medium for 72 h,and the protein expression of Opl1-ym Cherry was determined not to be increased due to the induction of oleic acid by Western-blot experiments and fluorescence observation techniques.In order to study the role of Opl1 p in lipid metabolism in cells and its effect on lipid droplet organelles,the OPL1 gene knockout Saccharomyces cerevisiae strain opl1Δ and OPL1 overexpressing yeast strain OPL1-OE were constructed in this experiment,and the detection of OPL1 knockout or In the case of overexpression,the effects of Saccharomyces cerevisiae on the growth status and lipid droplets of opl1Δ and OPL1-OE strains.Distribution has no effect.In order to realize the large-scale expression and purification of Opl1 p,using Saccharomyces cerevisiae,Pichia and Escherichia coli as hosts,an Opl1 p expression strain with GST or His(6)tag was constructed.It has been verified that the homologous protein expression Saccharomyces cerevisiae strain Opl1 p protein expression is low,and the heterologous protein expression Pichia pastoris strains,intracellular protein expression strains Opl1-GST and Opl1-His(6)can achieve large amounts of expression and purification,but The strain is unstable and cannot achieve stable and high protein expression.The heterologous protein expression Escherichia coli strains Opl1-GST and Opl1-His(6)can also achieve large-scale expression and purification,and the Opl1-His(6)was successfully purified and prepared by Ni-NTA affinity chromatography magnetic beads.Ultrafiltration and concentration yielded a higher purity protein.The substrate of Opl1 p was predicted by molecular docking technology,and it was found that the potential substrate of Opl1 p was monoacylglycerol.Combined with the results of homology analysis,it was determined that MAG was used as the substrate,and the Opl1-His expressed in E.coli was used(6)The enzyme activity analysis was carried out,and the experiment showed that Opl1-His(6)had no enzyme activity.