The Expression Optimization And Crystallographic Study Of Microcystinase-C

In recent years,cyanobacterial blooms frequently occur in eutrophic freshwater ecosystems worldwide as a result of climate change and increased nutrient loading.The cyanobacterial blooms not only cause bad effect for ecological environment,but also threaten human health because of the toxin second metabolites,just like microcystins(MCs)according to releasing into water via cell lysis.Our study aims to biodegrade microcystins by microcystinases which is characterised as a safe,efficient and high selectivity treatment.According to a series of Expression and purification of MlrC,we get purity and high protein by simple heterogenous recombination method.In order to elaborate the degrading mechanism of MlrC,we devote ourself to preliminary study research how to get the MlrC protein crystal with high diffraction.The detail research contents as follows:1.Optimization of the expression and purity of microcystinase MlrCTo get the soluble MlrC protein,we optimize the vector,competent cell,induction temperature,inductor concentration.Eventually,we find that the best expression condition are pET15d/pET21b vector,BL21(DE3)competent cell,22℃ induction temperature,200 μM inductor concentration.At the same time,we test the protein property by affinity chromatography,gel filtration chromatography,western blot and HPLC.Eventually we get high purity,high field,uniform property MlrC protein with biological activity finally.2.Analyzation of MlrC bioinformatics and structure predictionWe analyze the basic physicochemical properties,amino acid composition,secondary structure and tertiary structure prediction of MlrC protein.According to these analyzations,we can better optimize the unstable domain.3.Optimization of MlrC protein used for crystalIn order to stabilize the MlrC protein,we screen the methods from three aspects.The first is optimization of fusion tag,like the location of tag or without tag for full length MlrC.Owing to MlrC exists 6 cysteines,we mutate cysteine to serine.We also truncate the MlrC by N terminal truncation,C terminal truncation and internal delete.At last,we co-express the two domains of MlrC to get the more stable protein.4.Crystal screen and optimizatio of MlrCBased on the optimization of MlrC protein,we screen and optimization the crystallization condition.For crystal optimization,we try different precipitant,buffer solution,salty ions and additive screen.Finally we obtain the crystal which can be tested difraction.Because of no diffraction for initial crystal,we try some ways to optimize diffraction,such as Post-crystallization,transforming growth temperature,cryoprotectants screen and so on.Within these ways,we find that the cryoprotectants are the significant factor for improving diffraction.According to screen cryoprotectants and their working concentration,we get a optimal cryoprotectant(2-Methyl-2,4-pentanediol,MPD)which can prominently improve the diffraction to 2.6 A.