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首页> 外文期刊>Protein Expression and Purification >Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies
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Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

机译：从大肠杆菌包涵体中纯化具有酶活性的人赖氨酰氧化酶和类似赖氨酰氧化酶的蛋白

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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 pET21 a 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 0.150U/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. (C) 2003 Elsevier Inc. All rights reserved. [References: 26]
机译：赖氨酰氧化酶（LOX）是一种胞外铜依赖性酶，催化胞外基质蛋白中赖氨酸衍生的交联。最近的分子克隆表明存在由LOX和四个赖氨酰氧化酶样蛋白（LOXL; LOXL，LOXL2，LOXL3和LOXL4）组成的LOX家族。 LOX家族的每个成员都包含一个铜结合域，赖氨酰-酪氨酰醌残基和一个细胞因子受体样域。最近，新功能，如肿瘤抑制，细胞衰老和趋化性，已归因于该胺氧化酶家族，但家族成员之间的功能差异尚待确定。为了有效表达和纯化，我们将与LOX（LOX-p）和LOXL（LOXL-p1和LOXL-p2）的蛋白水解加工形式相对应的cDNAs克隆到细菌表达载体pET21a中，该载体具有6个连续的组氨酸密码子连接到3'基因的重组蛋白经镍螯合亲和层析纯化，并在N-月桂酰肌氨酸盐和Cu2 +存在下通过逐步渗析转化为酶促活性形式。纯化的LOX-p，LOXL-p1和LOXL-p2蛋白分别显示出比胺氧化酶活性为0.097、0.054和0.150U / mg，这被LOX的特异性抑制剂β-氨基丙腈（BAPN）抑制。这些纯净和活性形式的LOX和LOXL的可用性将大大有助于与该胺氧化酶家族的底物特异性和晶体结构有关的功能研究。 （C）2003 Elsevier Inc.保留所有权利。 [参考：26]

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