The Effect Of GAP Compound Preparation On The Expression Of E. Coli Recombinant Protein

E.coli is one of the most widely used hosts for the production of heterogonous proteins.Therefore,it is important to improve the efficiency of E.coli protein expression.Isopropyl-?-D-galactoside(IPTG)is an efficient inducer of expression vector T7 lactose promoter,but the high price and toxicity have restricted its massive use.As a cheap and non-toxic disaccharide,lactose can take place of IPTG as an inducer to a certain extent.However,there are still many deficiencies in protein expression due to the nature of lactose and the way in inducing,resulting in inefficiency of protein expression and low productivity.E.coli produces a variety of organic acids in the glycolysis pathway,which will ultimately affect cell viability and the expression of exogenous protein.In order to improve the expression of protein in a safe and effective way,this research is aimed at solving above problems of E.coli in exogenous protein.In order to obtain optimized GAP compound preparation,we use E.coli BL21 as the host bacteria and y-GGT as a reporter gene to improve the expression of y-GGT protease and enzyme activity.The main contents are:firstly,through single factor experiments,we investigate the effects of GAP,lactose,NA,the concentration of ions,amino acids and other factors on ?-GGT protease expression and host bacteria's growth;secondly,we explore the main effects and interaction of various single factor substances on y-GGT protease activity by Plackett-Burman experiment;thirdly,we optimize the GAP compound components with Box-Behnken experiment;fourthly,we test the effects of GAP compound on other exogenous protein's expression,and experiment in 6L fermentor.Main results are as follows:1.By the single factor experiment of y-GGT protease expression,we studied the influences of lactose,GAP,NA,NH4+,Mg2+,Gin,Ser,Arg,and others on y-GGT protease expression.The result shows that GAP plays a regulatory role on the metabolism of host bacteria as modifier and carbon.It could promote the expression of y-GGT protein and cell growth,and the optimal concentration is 5 g/L.Adding 0.3 g/L NA is beneficial to the generation of intracellular NAD(H)and affects the state of cells and promotes the expression of target protein.Ions especially NH4+ and Mg2+ mainly impact the y-GGT protease activity.1.5 g/L Gin and 1.5 g/L Ser benefit the y-GGT protein's expression and the growth of host bacteria well.The effects of other amino acids are not obvious.2.Selecting eight factors,we investigated the main effects and interaction of various single factor substances on ?-GGT protease's activity by PB experiment.The result shows lactose,GAP and NA influence ?-GGT protease activity significantly.3.The best combination of GAP compound preparation is lactose 2.3 g/L,GAP 5.6 g/L,NA 0.28 g/L through Box-Behnken experiment,which could improve the protease activity value to 98.6 U/L,increased by 13.3%when compared with non-optimized control group.Additionally,the optimized GAP compound preparation improves the density of bacteria and protein expression than the control group significantly.4.Through the validation experiments,we find that GAP compound preparation promotes the expression of foreign proteins in E,coil significantly,which is better than the induction of IPTG.In 6 L fermenter amplification experiment,GAP compound promoted the growth of cell significantly,and OD600 reached 15.8.The density of bacteria doubled compared with the IPTG control group.The percentage of y-GGT protease in total protein increased by 19.4%compared with the control group and enzyme activity of per unit volume increased by 90%,about 1032 U/L.Compared with the control group,the crude ?-GGT solution induced by GAP-compound is more beneficial for the theanine production.GAP-compound can not only promote cell growth and enhance the expression of the target protein significantly,but also replace toxic IPTG as en effective inducer.It is safe,efficient and cheap,which will provide new methods and ideas for large-scale microbial fermentation,production and preparation of enzyme protein and has profound influences on the fermentation industry.