Vector and method for expressing L-aspartic acid-alpha-decarboxylase by recombinant Escherichia coli

[0104] Example 1:

[0105] 1) Comparative culture of double-plasmid co-expression strain pET32a-pCDFDuet-ADC-BL21(DE3) and single-plasmid strain pET32a-ADC-BL21(DE3) shake flask cell fermentation

[0106] Co-express pET32a-pCDFDuet-ADC-BL21(DE3): Glycerol tube 100μL → 10mL test tube LB liquid medium (add 100mg / L ampicillin and 50mg / L streptomycin), 30°C, 150RPM overnight culture, transfer 5mL → 500mL TB shake flask fermentation medium filling volume 200mL (add 100mg / L ampicillin and 50mg / L streptomycin), 37 ° C, 180RPM for 6 hours → add 10g / L α-lactose induction, induction temperature 30°C→After 17 hours of induction, centrifuge to collect cells→Take 1g of centrifuged wet cells and add 10g of deionized water to mix well→Sonicate and centrifuge, take the crushed supernatant and crushed solution to precipitate, add deionized water to dilute the reset solution ten times, and run proteins separately Electrophoresis SDS-PAGE detection protein expression.

[0107]pET32a-ADC

[0112] Example 2:

[0113] 1) Comparative culture of double-plasmid co-expression strain pET32a-pCDFDuet-ADC-BL21(DE3) and single-plasmid strain pCDFDuet-ADC-BL21(DE3) shake flask cell fermentation

[0114] pET32a-pCDFDuet-ADC-BL21(DE3): Glycerol tube 100μL→10mL test tube LB liquid medium (add 100mg / L ampicillin and 50mg / L streptomycin), 30℃, 150RPM overnight culture, transfer 5mL→500mL The volume of the TB shake flask fermentation medium is 200mL (add 100mg / L ampicillin and 50mg / L streptomycin), 37°C, 180RPM for 6 hours → add 10g / L α-lactose induction, induction temperature 30°C → After 17 hours of induction, centrifuge to collect cells → Take 1g of centrifuged wet cells and add 10g of deionized water to mix them → Ultrasonic crushing and centrifugation, take the crushed supernatant and the precipitate of the crushed liquid, add deionized water to dilute the reset solution ten times, and run protein electrophoresis SDS-PAGE detection Protein expression.

[0115] pCDF

[0119] Example 3:

[0120] 1) The three-plasmid co-expression strain pET28a-pET32a-pCDFDuet-ADC-BL21(DE3) and the double-plasmid strain pET32a-pCDFDuet-ADC-BL21(DE3) were compared and cultured in shake flask cells

[0121] Two-plasmid co-expression of pET32a-pCDFDuet-ADC-BL21(DE3): Glycerol tube 100μL → 10mL test tube LB liquid medium (add 100mg / L ampicillin and 50mg / L streptomycin), 30°C, 150RPM overnight culture, transfer Connect 5 mL → 500 mL of TB shake flask fermentation medium with a liquid volume of 200 mL (add 100 mg / L ampicillin and 50 mg / L streptomycin), 37 ° C, 180 RPM for 6 hours → add 10 g / L α-lactose to induce, Induction temperature at 30°C → End of induction for 17 hours and centrifuge to collect cells → Take 1g of centrifuged wet cells and add 10g of deionized water to mix well → Ultrasonic crushing and centrifugation, take the crushed supernatant and crushed liquid to precipitate, add deionized water to dilute the reset solution ten times, respectively