Study On Fermentation And Purification Optimization Of Recombinant T4 DNA Ligase

T4 DNA ligase is a key tool enzyme responsible for DNA connection.T4 DNA ligase is a 55.23 kda single strand polypeptide(Gen Bank:X00039.1),which catalyzes the formation of phosphodiesteryl bond on the adjacent 3-hydroxy and 5-phosphate ends of double strand DNA.The traditional purification method of T4 DNA ligase recombinant protein includes obtaining crude enzyme solution.PEI precipitation,ammonium sulfate precipitation,molecular sieve chromatography,DE-52 column chromatography,nickel column method and so on.However,it is not suitable for large-scale industrial production due to its complicated operating procedures,high cost and small purification volume.With the development of protein purification technology,the combination of various purification methods has become a tentative research direction.In this paper,nickel column and ion column were combined for purification.The results showed that large-scale purification can be achieved by the combination of nickel column and ion column.The T4 DNA protease obtained has high purity and stable activity.The purpose of this paper is to study the optimization of fermentation and purification conditions of T4 DNA ligase,and to apply it to industrial production.In this paper,T4 DNA ligase gene was obtained by molecular cloning method,and the recombinant expression plasmid pet15b-T4 was constructed.After chemical transformation,the recombinant expression plasmid was transferred into the host BL21(DE3)to induce expression,and the host bacteria was screened to obtain a high-yield and stable recombinant engineering bacteria,named BL21(DE3)-15b-T4.In order to meet the needs of stable production in industry,a strain bank was established and its stability was explored,including the morphology and biochemical characteristics of the strain.The results showed that the recombinant bacteria had typical morphological and physiological characteristics of Escherichia coli.After sequencing and protein gel detection,the target gene did not mutate,and after one year of preservation,the stability and expression of the strain meet the needs of productionIn this study,the conditions of shake flask fermentation,the exploration and optimization of 50L fermentor fermentation technology,and the exploration and optimization of protein purification technology were studied.Through the optimization of induction time,concentration and temperature of IPTG,the final concentration of IPTG was 0.5mm and induction temperature was 25? at 1% inoculum amount,and the highest amount of target protein was achieved at 12 hours induction,which provided technological reference for subsequent fermentation.On this basis,the fermentation conditions of 50L fermentor are further optimized.Under the optimized fermentation conditions,3kg of bacteria can be obtained from 50L fermentor.After preliminary detection,it contains 20mg of crude protein/g of bacteria.The specific fermentation conditions are as follows:the best inoculation time&inoculation amount:the first stage seed culture time is 10-12 h,the second stage seed culture time is 3-4 h.The best inoculation amount was 4%.The best medium formula:glucose 11.7g/L,peptone 3.2 g/L,yeast 1.8 g/L,potassium dihydrogen phosphate trihydrate 11 g/L,potassium dihydrogen phosphate 3g/L,ammonium citrate 4 g/L,magnesium sulfate heptahydrate 1.2 g/L.The best specific growth rate was 0.2-0.3 h^-1before induction and 0.07h^-1after induction.The best inducer condition was IPTG 0.5 m M,OD₆₀₀20 and temperature25?.Fermentation end point:12 hours after induction,if the OD of bacteria is not growing,it can be treated in the tank.Using 300L fermentation tank for fermentation amplification,the scale-up of small-scale fermentation process was successfully realized.All indicators were consistent with the level of 50 L tank.The batch yield of bacteria could reach 15 kg,and the yield increased by more than 5 times.In addition,based on the existing basic purification conditions,the pilot scale-up of purification process was carried out,and the yield was increased by 10 times.Repeat three batches and pass QC inspection.It laid a technological foundation for further expanding the preparation scale in the future.The results showed that under the optimized purification conditions,the purified T4 DNA ligase 2 g could be obtained.The purity of T4 DNA ligase was more than 95%,and the activity of T4 DNA ligase obtained by large-scale purification is500 U/?L,which meets the storage conditions and has good stability between batches.In this paper,a recombinant BL21(DE3)-15b-T4 was successfully constructed,which can be used to produce T4 DNA ligase on a large scale.The fermentation technology and amplification purification technology of the fermentation tank were explored and optimized.Finally,a high expression of T4 DNA ligase and a stable T4 DNA ligase were obtained,realizing industrial production and application.