The expression of spinach glycolate oxidase (GO) in E. coli and the application of GO in the production of glyoxylic acid
The complementary deoxyribonucleic acid (cDNA) coding spinach glycolate oxidase (GO) was amplified by reverse transcription polymerase chain reaction (RT-PCR), using the total ribonucleic acid (RNA) of spinach leaves as the template, and was cloned into cloning vector pMD18-T. After the DNA sequence was determined, the go gene was subcloned into Escherichia coli expression vectors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that recombinant GO was expressed in E. coli BL21 (DE3)(pTIG-Trx-GO) and E. coli BL21 (DE3)(pET-22b(+)-GO). The result of the enzyme activity assay and the oxidation of glycolate to glyoxylate catalyzed by the whole cells of E. coli BL21 (DE3)(pET-22b(+)-GO) proved the feasibility of using E. coli cells to produce glyoxylic acid.
Index EntriesGlycolate oxidase glyoxylic acid cloning expression
Unable to display preview. Download preview PDF.
- 1.Anton, D. and DiCosimo, R. (1994) Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase. Patent WO 94/06925, 31 March, 3–4.Google Scholar
- 5.Anton, D. and DiCosimo, R. (1995) Glycolate oxidase production. Patent WO 95/01444, 12 Jan, 5–8Google Scholar
- 9.Wood C, D. M. et al. (1989) Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 16, 3469–3478.Google Scholar
- 12.Zhao, Z. H., Zhang, Y. S., Liu, C. X., and Ma, Q. J. (2000) A new strategy for soluble expression of foreign proteins in E. coli Biotechnol. Lett. 11, 164.Google Scholar
- 14.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
- 15.Ausubel, F. M. (1995) Current Protocols in Molecular Biology, 3rd ed., Publishing Associates and Wiley Interscience, New York.Google Scholar
- 17.Volokita, M. and Somervillet, R. C. (1987) The primary structure of spinach glycolate oxidase from the DNA sequence of a cDNA clone. J. Biol. Chem. 262, 15,825–15,828.Google Scholar