| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 69861 | Journal of Molecular Catalysis B: Enzymatic | 2012 | 6 Pages |
In this work, a reliable protocol was designed to rapidly express and purify a rat diamine oxidase in Escherichia coli as a useful alternative to enzyme isolated from animal sources. The cDNA encoding for rat diamine oxidase was overexpressed in an Origami2(DE3) E. coli strain and, by employing a rapid purification protocol in which the hexahistidine tag was added at the C-terminal end of the enzyme, the recombinant oxidase could be purified in a single step on a Ni-NTA column at >95% purity. The enzyme was active but was largely produced in an immature quinone form: Cu2+ ions stimulated further activation/maturation. This expression and purification procedure offers an easy and rapid means of producing recombinant rat diamine oxidase from an animal-free source and represents a useful tool to boost biotechnological application of this enzyme.
Graphical abstract.Figure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Rat diamine oxidase (DAO) was overexpressed in E. coli. ► The purified recombinant DAO (2 mg pure protein/L broth) was ≥95% pure. ► The recombinant DAO was active but was largely produced in an immature quinone form. ► Cu2+ ions significantly stimulated further activation/maturation. ► This procedure produces recombinant rat diamine oxidase from an animal-free source.
