Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

Sang Taek Jung, Moon Suk Kim, Ji Yeon Seo, Hyung Chul Kim, Youngho Kim

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3′ of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu2+. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by β-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.

Original languageEnglish
Pages (from-to)240-246
Number of pages7
JournalProtein Expression and Purification
Volume31
Issue number2
DOIs
Publication statusPublished - 2003 Jan 1
Externally publishedYes

Fingerprint

Protein-Lysine 6-Oxidase
Escherichia coli Proteins
Inclusion Bodies
Amines
Oxidoreductases
Copper
Aminopropionitrile
Cytokine Receptors
Cell Aging
Extracellular Matrix Proteins
Molecular Cloning
Chemotaxis
Substrate Specificity
Nickel
Affinity Chromatography
Recombinant Proteins
Histidine
Codon
Lysine
Dialysis

Keywords

  • Amine oxidase
  • Lysyl oxidase
  • Lysyl oxidase-like protein
  • Refolding

ASJC Scopus subject areas

  • Biotechnology

Cite this

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies. / Jung, Sang Taek; Kim, Moon Suk; Seo, Ji Yeon; Kim, Hyung Chul; Kim, Youngho.

In: Protein Expression and Purification, Vol. 31, No. 2, 01.01.2003, p. 240-246.

Research output: Contribution to journalArticle

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