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.
2012
Amine oxidase; Animal-free enzyme; Amines assay
File in questo prodotto:
File Dimensione Formato  
2012 MolCatalB.pdf

Accesso riservato

: Post-Print (DRAFT o Author’s Accepted Manuscript-AAM)
Dimensione 541.88 kB
Formato Adobe PDF
541.88 kB Adobe PDF   Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11311/676362
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 5
  • ???jsp.display-item.citation.isi??? 6
social impact