title: Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies.
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 beta-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.
authors.affiliation: Laboratory of Functional Proteomics, Samyang-Genex Biotechnology Research Institute, 63-2, Hwaam-Dong, Yusung-Gu, Daejeon City, Republic of Korea.
authors.forename: Sang Taek
authors.initials: ST
authors.lastname: Jung
authors.forename: Moon Suk
authors.initials: MS
authors.lastname: Kim
authors.forename: Ji Yeon
authors.initials: JY
authors.lastname: Seo
authors.forename: Hyung Chul
authors.initials: HC
authors.lastname: Kim
authors.forename: Youngho
authors.initials: Y
authors.lastname: Kim
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001596:Benzylamines / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D005612:Freeze Drying; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D015202:Protein Engineering; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001596:Benzylamines; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C445313:LOXL1 protein, human; C119091:LOXL2 protein, human; D008249:Protein-Lysine 6-Oxidase; D006631:Amine Oxidase (Copper-Containing)