title: Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility.
abstract: Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM(-1 )cm(-1). No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture.
authors.affiliation: MI-SWACO, Shafter, CA 93263, USA.
authors.forename: Madison A
authors.initials: MA
authors.lastname: Smith
authors.affiliation: University of California, San Francisco, San Francisco, CA 94143, USA.
authors.forename: Jesica
authors.initials: J
authors.lastname: Gonzalez
authors.affiliation: California State University, Bakersfield, Bakersfield, CA 93311, USA.
authors.forename: Anjum
authors.initials: A
authors.lastname: Hussain
authors.affiliation: California State University, Bakersfield, Bakersfield, CA 93311, USA.
authors.forename: Rachel N
authors.initials: RN
authors.lastname: Oldfield
authors.affiliation: California State University, Bakersfield, Bakersfield, CA 93311, USA.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Johnston
authors.affiliation: California State University, Bakersfield, Bakersfield, CA 93311, USA.
authors.forename: Karlo M
authors.initials: KM
authors.lastname: Lopez