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Cloning and coexpression of recombinant N-demethylase B and Glycolate oxidase genes in Escherichia coli
- Li, Dengchao, Han, Qiumin, Zhang, Tong
- Molecular biology reports 2019 v.46 no.1 pp. 505-510
- (S)-2-hydroxy-acid oxidase, Escherichia coli, Pseudomonas putida, Spinacia oleracea, affinity chromatography, ampicillin, genes, genetic vectors, molecular cloning, molecular weight, nickel, plasmids, polyacrylamide gel electrophoresis, screening, sodium dodecyl sulfate, spinach
- NdmB genes from Pseudomonas putida CBB5 and GO genes from spinach, which encode N-demethylase B (NdmB) and Glycolate oxidase (GO) respectively, were separately ligated into expression vectors of pACYCDuet-1 and pET32a to construct recombinant plasmids of pACYCDuet-1-ndmBHis (pBH) and pET32a-GOHis (pGOH). Then the two plasmids were both transformed in Escherichia coli (E. coli) strain BL21 (DE3) and screening the recombinants (pBHGOH) using ampicillin and chloramphonicol as two antibiotics in Luria–Bertani medium. After induction with IPTG, both recombinant ndmB and GO genes were coexpressed in E. coli. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) showed that the estimated molecular weight of NdmB and GO was 35 kDa and 40 kDa, respectively. By two-step purification of Ni affinity chromatography and Q-Sepharose chromatography, the coexpressed NdmB and GO were separated and resulted in a 15.8-fold purification with 8.7% yield and 12.8-fold purification with 7.2% yield, respectively.