Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility.

Madison A Smith,Anjum Hussain,Rachel N Oldfield,Kathryn A Johnston,Karlo M Lopez,Jesica Gonzalez

doi:10.1155/2016/5098985

Madison A Smith, Anjum Hussain + Show 4 more

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https://doi.org/10.1155/2016/5098985

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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.

Highlights

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that catalyzes a key cross-linking step in collagen and elastin [1, 2]
All PCR materials were obtained from New England Biolabs (NEB) with the exception of the primers which were purchased from Midland Certified Reagent Company and the polymerase which was purchased from Agilent
The DNA cassette corresponding to the mature form of lysyl oxidase, starting at Asp 169, was successfully isolated from plasmid pLOX02 using PCR

Summary

Introduction

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that catalyzes a key cross-linking step in collagen and elastin [1, 2]. Aside from its structural role in the extracellular matrix, LOX has been implicated as playing a role in other biological processes including morphogenesis and repair of connective tissues, developmental control, and chemotaxis [2, 4, 5]. LOX is dependent on two cofactors: a tightly bound Cu(II) atom [9], which is required for the posttranslational modification of Tyr 355 to form the second cofactor, lysyl tyrosyl quinone (LTQ) [10, 11]. LTQ is formed by the cross-linking of Tyr 355 with Lys 320 to form the catalytic site of the enzyme. It has been postulated that the copper atom is required for the catalytic activity of the enzyme [1, 9]; this postulate has been disputed [12]

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