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.
Expression of rat diamine oxidase in Escherichia coli
D'ARRIGO, PAOLA;
2012-01-01
Abstract
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.File | Dimensione | Formato | |
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