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Gene Synthesis> | To construct the expression vector, a fragment containing promoter PrpoS, cbm3 (family 3 carbohydrate-binding module from Clostridium thermocellum) coding sequence, and T7 terminator chemically synthesized by GenScript (Nanjing, China) was inserted into pUC57 previously digested with NdeI/HindIII. The resulting construct was named pRPOCB. Two primers (5′-GAATTCCTCGAGGGCTCTTCCAG-3′, 5′-GGATCCCGGTTCTTTACCCCAAACC-3′) were used for PCR to generate the linearized vector sequence, and two other primers (5′-GCCCTCGAGGAATTCTTAGTGGGTAAAGCCGTACTTTTTCAGG-3′, 5′-AAAGAACCGGGATCCATGTCACTTTACGGAAAGTACGACCAAG-3′) were used to amplify gadB gene. The two PCR products were assembled into a recombinant plasmid, generating pRPOCB-gadB, using In-Fusion HD Cloning Kit (Takara Biomedical Technology Co., Beijing, China); finally, the vector was transformed into E. coli DH5α competent cells. | Get A Quote |
Background GABA (γ-aminobutyric acid) is a four-carbon nonprotein amino acid that has hypotensive, diuretic, and tranquilizing properties. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple and economical method of preparing and immobilizing GAD would be helpful for GABA production. In this study, the GAD from Lactobacillus fermentum YS2 was expressed under the control of a stress-inducible promoter and was purified and immobilized in a fusion form, and its reusability was investigated. Results The fusion protein CBM-GAD was expressed in Escherichia coli DH5α carrying pCROCB-gadB, which contained promoter PrpoS, cbm3 (family 3 carbohydrate-binding module from Clostridium thermocellum)... More