Cloning,Expression And Preliminary Study Of Oligosaccharide Binding Protein From Lactobacillus Acidophilus

Lactic acid bacteria are closely related to human health.Increasing the colonization of lactic acid bacteria in intestinal tract is conducive to maintaining the stability of intestinal microecological environment.Oligosaccharides can't be used by human body and reach intestinal tract directly after being ingested by human body.Some researches have found that oligosaccharides can be used by lactic acid bacteria in intestinal tract and promote the proliferation of lactic acid bacteria.But how do lactic acid bacteria utilize oligosaccharides? Whether there are some oligosaccharide binding proteins involved in oligosaccharide transport is still unclear.The main content of this study is to construct two in vitro expression strains of Lactobacillus acidophilus oligosaccharide binding protein,explore the soluble expression conditions of the protein and obtain high-purity target protein,and preliminarily explore the crystal growth conditions to lay a foundation for studying the mechanism of oligosaccharide binding protein participating in oligosaccharide transportation.In this study,two oligosaccharide binding proteins LaMsmE and LaMsmE?were analyzed by bioinformatics to understand the primary structure and basic physiological properties of the proteins,which are hydrophilic s Tab.proteins.Through signal peptide prediction and transmembrane region prediction,it is predicted that these two proteins are both receptor proteins that play a signaling role in cytoplasm.Domain analysis found that they all have the same independent domains SBP?bac?1 and SBP?bac?8.The two proteins were used to construct a protein evolutionary tree and motif analysis.It was found that the homology of protein LaMsmE and protein LaMsmE? is 8%,and there is a distant relationship,but they all have motif 1.It is speculated that they originate from the same ancestor,but many different changes have occurred in the evolution process.In the early stage of this study,the insoluble expression of full-length LaMsmE protein was tested.After that,according to the bioinformatics analysis of the protein LaMsmE,the amino acid fragments at positions 17-431 were intercepted for research.LaMsmE gene was successfully cloned from Lactobacillus acidophilus genome and recombinant plasmid was successfully constructed with vector p ET-28a-MBP.The soluble expression of the fusion protein MBP-LaMsmE was tested under two induction conditions.The fusion protein showed a large amount of soluble expression at 22?,0.5 m M IPTG.The fusion protein MBP-LaMsmE was cleaved by TEV protease.The optimal enzyme digestion conditions were obtained by exploring the enzyme digestion time and the dosage of protease.The protein sample under the optimal enzyme digestion conditions was purified by ion exchange chromatography,and the LaMsmE protein with a purity as high as 95% was obtained.This study has solved the problem that the full-length LaMsmE protein is difficult to be soluble expressed,and laid a foundation for further research on the structure and function of the protein.In this study,amino group 36-415 of LaMsmE? was intercepted through bioinformatics analysis of the protein.LaMsmE? gene was successfully cloned from Lactobacillus acidophilus genome and recombinant plasmid was successfully constructed with vector p ET-15 b.The soluble expression of protein LaMsmE? was tested under two induction conditions.the protein showed a large amount of soluble expression at 30?,0.05 m M IPTG.In order to analyze the effect of protein on oligosaccharide from protein structure,the growth conditions of protein crystals were preliminarily screened.In order to determine the effect of key amino acid residues of protein on oligosaccharides,based on amino acid electrification and previous studies,the site of action of quasi-key amino acid residues of protein LaMsmE? was predicted,and three mutant plasmids were successfully constructed through gene site-directed mutation.This study provides conditions for studying the mechanism of oligosaccharide binding protein LaMsmE? involved in oligosaccharide transportation,lays a foundation for clarifying the mechanism of Lactobacillus acidophilus using oligosaccharide,and provides reference basis for developing functional foods with sui Tab.prebiotics and probiotics combination..