Construction Of Human Cu,zn-SOD Engineering Strains Of Escherichia Coli And Saccharomyces Cerevisiae And Optimization Of Fermentation Conditions

Production of human Cu,Zn-SOD by genetic engineering technology has the advantages of high yield,stable activity and high purity.Moreover,this method avoids the immune allergic reaction of Cu,Zn-SOD heterologous protein rejection and solves the problem of extraction of Cu,Zn-SOD from blood.It is of great significance to the industrial production of Cu,Zn-SOD and its application in medical,biochemical and food products.In this study,we used genetic engineering methods to construct prokaryotic expression engineering bacteria and eukaryotic expression engineering bacteria of human Cu,Zn-SOD.The fermentation conditions of the two engineering strains were optimized to increase expression levels and enzyme activity,and the two expressed engineered bacteria were compared and analyzed to facilitate subsequent research and production.In this study,the nucleotide sequences of human Cu,Zn-SOD were modified and synthesized artificially based on Escherichia coli?E.coli?codon bias,and were combined with plasmid pET-22b?+?to construct an expression vector and transformed into E.coli BL21?DE3?,Secreted expression was induced by IPTG,and its fermentation conditions were further optimized to increase the expression level and protein activity.Based on the codon bias of Saccharomyces cerevisiae?S.cerevisiae.?,the nucleotide sequence of human Cu,Zn-SOD was modified and synthesized artificially.The expression vector was constructed by combining it with plasmid pAUR123 and transformed into S.cerevisiae.The fermentation conditions were further optimized to improve expression levels and protein activity.Through the use of the dominant codons of engineering bacteria to transform the foreign target genes,increase the heterologous expression levels of the engineered bacteria,give full play to their respective advantages,and tap the production potential of E.coli and S.cerevisiae as heterologous expression engineering bacteria for subsequent research and industrialization.Lay the foundation for production.The main contents of this study are as follows:?1?The nucleotide sequence of human Cu,Zn-SOD was modified based on E.coli codon bias,combined with plasmid information to add restriction enzyme EcoR I and Hind III restriction sites at both ends of the sequence,and synthesized the target gene sequence Cu,Zn-SOD-E.coli was ligated with Cu,Zn-SOD-E.coli and plasmid pET-22b?+?by double digestion with T4 ligase to construct the expression vector pET-22b-SOD.Transformation into E.coli BL21?DE3?,construction of human Cu,Zn-SOD engineering strains of E.coli named BL21-22b-SOD.?2?E.coli engineering bacteria BL21-22b-SOD was cultured and induced by IPTG.The protein expression of the engineered bacteria was detected by SDS-PAGE and Bradford method.The results showed that the molecular weight of the target bacteria expressed by BL21-22b-SOD was approximately At 16 kDa,the total protein concentration was 19.91 mg/mL,the target protein was approximately 17.6%of the total protein,and the target protein concentration was 3.50 mg/mL.The crude enzyme activity was determined by pyrogallol autoxidation method.The activity of the crude enzyme solution was 977.2 U/mL.The calculated specific activity was 279.2 U/mg.?3?Based on the nucleotide sequence of human Cu,Zn-SOD and S.cerevisiae codon bias,combined with plasmid information,the restriction endonuclease Sma I and Sac I restriction sites were added at both ends of the sequence and synthesized.The target gene sequence Cu,Zn-SOD-Yeast was ligated with Cu,Zn-SOD-Yeast and plasmid pAUR123 by double digestion with T4 ligase to construct expression vector pAUR123-SOD and transformed into S.cerevisiae to construct human Cu,Zn-SOD engineering strains of S.cerevisiae named Yeast-123-SOD.?4?Yeast-123-SOD expression was cultured in S.cerevisiaev and the expression of target protein was detected by SDS-PAGE and bradford method.The result showed that the molecular weight of the target strain Yeast-123-SOD was about 16 kDa.The total protein concentration was 20.18 mg/mL,the target protein was approximately4.7%of total protein,and the target protein concentration was 0.947 mg/m L.The activity of crude enzyme was measured by pyrogallol auto-oxidation method.The enzyme activity of crude enzyme was 285.5 U/m L.The calculated specific activity was 301.5 U/mg.?5?The fermentation conditions of the engineering bacteria BL21-22b-SOD were optimized from the initial induction of OD₆₀₀ value,the final concentration of IPTG,induction time,and induction temperature,and the optimum fermentation conditions were determined as:the initial induction of OD₆₀₀ of the bacteria was 0.8.The final concentration of inducer IPTG was 0.1 mmol/L,the induction time after induction was 6 hours,and the induction temperature was 35°C.The engineering bacteria were cultured under this condition,and the total protein content in the fermentation was determined by Bradford method.The results were analyzed by Bandscan analysis software.The percentage of target protein expression in total protein was 22.2%,and the target protein was obtained.The content of 6.5 mg/m L,enzyme activity of the crude enzyme was 2430.8 U/mL,and the enzyme activity was 375.2 U/mg.?6?The fermentation conditions of the engineering bacteria Yeast-SOD-123 were optimized from four aspects of fermentation time,fermentation temperature,initial pH,and dissolved oxygen content of the engineering bacteria.The optimum fermentation conditions were as follows:Fermentation dissolved oxygen at 36 hours,fermentation temperature 28°C,initial pH 5.5,and culture rotation speed at 200 rpm.The engineered bacteria were cultured under this condition,and the total protein content in the fermentation was determined by Bradford method.The SDS-PAGE protein was electrophoresed.The results were analyzed by Bandscan analysis software.The percentage of target protein expressed in total protein was 6.02%.The protein content was 1.505 mg/mL,the enzyme activity was 678.6 U/m L,and the specific enzyme activity was 450.9 U/mg.