Using Thioredoxin Solubility Tag in the Characterization of Lysyl Oxidase


Anjum Hussain


Karlo Lopez, Assistant Professor of Biochemistry, California State University, Bakersfield

Lysyl oxidase (LOX) is a copper-dependent enzyme that activates the formation of crosslinkages in collagen and elastin in connective tissues by oxidative deamination of lysine and hydroxylysine residues. LOX requires one covalently bound cofactor, lysyl tyrosyl quinone, and a copper ion in order to carry out catalysis. Currently, there is no crystal structure available for mammalian LOX, primarily due to the low solubility of the enzyme in aqueous buffers. The only crystal structure available for “lysyl oxiadase” is from Pichia pastoris which uses a different catalytic cofactor. In order to overcome the low solubility of lysyl oxidase, a thioredoxin solubility tag has been engineered on the N-terminus of mature mammalian LOX in order to facilitate the elucidation of the crystal structure of lysyl oxidase. Overexpresion of this enzyme yielded 4.4 mg of enzyme per liter of media. This is nearly double the yield obtained using the much larger Nus-A solubility tag suggesting that this tag has less of an effect on overexpression. The enzymatic activity for this tagged enzyme appears to be unaffected by the tag. LTQ content will be quantified by detection of a phenylhydrazone adduct via visible spectroscopy and the percent copper incorporation will be also determined. Once these parameters are established, crystallographic screens will be carried out in order to determine crystallography conditions leading to the elucidation of a crystal structure.

Presented by:

Anjum Hussain


Saturday, November 23, 2013




Poster Session 3 - Villalobos Hall

Presentation Type:

Poster Presentation