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

Q2 Biochemistry, Genetics and Molecular Biology

Enzyme Research Pub Date : 2016-01-31 DOI:10.1155/2016/5098985

Madison A Smith, J. Gonzalez, Anjum Hussain, Rachel N. Oldfield, Kathryn A. Johnston, Karlo Lopez

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引用次数: 6

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.

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利用溶解度标签过表达可溶性重组人赖氨酸氧化酶:对活性和溶解度的影响

赖氨酸氧化酶是一种重要的细胞外基质酶，由于其溶解度低，尚未被充分表征。为了克服该酶的低溶解度，在成熟赖氨酸氧化酶的n端设计了3个溶解度标签(Nus-A, Thioredoxin (Trx)， Glutathione-S-Transferase (GST))。总酶产率测定为:Nus-A标记的酶为1.5 mg (0.75 mg/L培养基)，Trx标记的酶为7.84 mg (3.92 mg/L培养基)，GST标记的酶为9.33 mg (4.67 mg/L培养基)。计算出us- a标记的酶的酶活性为0.11 U/mg, Trx标记的酶的酶活性为0.032 U/mg，而GST标记的酶没有检测到酶活性。所有三种溶解度标记形式的酶都含有铜;然而，GST标记的酶似乎比其他两种形式更有亲和力地结合外源性铜。使用先前报道的消光系数15.4 mM−1 cm−1，对Nus-A和Trx标记的赖氨酸氧化酶的催化辅助因子赖氨酸酪氨酸醌(LTQ)测定为92%。GST标记赖氨酸氧化酶未检测到LTQ。鉴于这些数据，看来Nus-A是从大肠杆菌培养中获得可溶性和活性重组赖氨酸氧化酶的最合适的标签。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Enzyme Research Biochemistry, Genetics and Molecular Biology-Biochemistry

CiteScore

4.60

自引率

0.00%

发文量