Cloning and coexpression of recombinant N-demethylase B and Glycolate oxidase genes in Escherichia coli
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.
KeywordsN-Demethylase B Glycolate oxidase Pseudomonas putida CBB5 Spinach Cloning Coexpression
This research was supported by Top-notch Academic Programs Project of Jiangsu Higher Education Institutions under Grant Number PPZY2015A018.
Compliance with ethical standards
Conflict of interest
The author declares that they have no conflict of interests.
- 1.Gopishetty SR, Louie MT, Yu CL, Subramanian MV (2011) Microbial degradation of caffeine, methylxanthines and its biotechnological applications. In: Thatoi HN, Mishra BB (eds) Microbial biotechnology: method and applications. Alpha Sci Int Ltd. Press, Oxford (UK), pp 44–67Google Scholar
- 11.Macheroux P, Mulrooney SB, Williams JrCH, Massey V (1992) Direct expression of active spinach glycolate oxidase in Escherichia coli. Biochem Biophys Acta 1132:11–16Google Scholar
- 13.De Sena AR, de Assis SA, Branco A (2011) Analysis of theobromine and related compounds by reversed phase high performance liquid chromatography with ultraviolet detection: an update (1992–2011). Food Technol Biotechnol 49:413–423Google Scholar