Production of recombinant human α1-microglobulin and mutant forms involved in chromophore formation
Production of recombinant human α1-microglobulin and mutant forms involved in chromophore formation
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DOI：
10.1016/j.pep.2006.10.023
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发表时间：
2007-05-01
影响因子：
1.6
通讯作者：
Wasylewski, Zygmunt
中科院分区：
文献类型：
--
作者：
Kwasek, Anna;Osmark, Peter;Wasylewski, Zygmunt
α-微球蛋白是一种存在于血浆和组织中的26 kDa脂质运载蛋白，携带一组未知的发色团，与C34、K92、KI 18和KI 30结合，这导致其电荷和大小异质性。在人类中，该蛋白质以两种形式存在，全长和缺乏C-末端四肽LIPR（t-alpha（1）-微球蛋白），两者都是血红素结合的，后者具有血红素降解特性。我们报道了在大肠杆菌中克隆和过表达全长α 1-微球蛋白（wt蛋白）、t-α 1-微球蛋白（wtz LIPR）和突变体C34 S、K（92，118，130）T和C34 S/K（92，118，130）T，后者随后缩写为K（3）T和C34 S/K（3）T。经纯化和复性后的包涵体，所有的蛋白质正确折叠的远紫外圆二色谱和放射免疫测定法确定。如凝胶过滤所示，重组α（1）-微球蛋白形成二聚体的趋势低于人血浆或尿液类似物。所有α 1-微球蛋白形式的发色团含量均高于牛血清白蛋白，但显著低于人尿液或血浆对应物。吸光度和荧光曲线的差异与发色团通过与血红素或其他发色团前体的一系列反应形成的模型一致，并且其中C34对于配体的结合是必需的，K92、KI 18和K130参与向发色团的转化，并且LIPR抑制后一反应。(c)2006年爱思唯尔公司All rights reserved.
alpha-Microglobulin, a 26 kDa lipocalin present in plasma and tissues, carries a set of unknown chromophores, bound to C34, K92, KI 18 and KI 30, which cause its charge and size heterogeneity. In man, the protein is found in two forms, full length and lacking the C-terminal tetrapeptide LIPR (t-alpha(1)-microglobulin), both which are heme-binding and the latter with heme-degrading properties. We report cloning and overexpression of full length alpha(1)-microglobulin (wt protein), t-alpha(1)-microglobulin (wtz LIPR) and the mutants C34S, K(92,118,130)T and C34S/K(92,118,130)T, the latter subsequently abbreviated as K(3)T and C34S/K(3)T, in Escherichia coli. After purification and refolding from inclusion bodies, all proteins were correctly folded as determined by far-UV circular dichroism and radioimmunoassay. As revealed by gel filtration, recombinant alpha(1)-microglobulins had lower tendencies to form dimers than human plasma or urine analogues. All alpha(1)-microglobulin forms displayed higher amounts of the chromophore than bovine serum albumin but significantly lower than the human urine or plasma counterparts. Differences in the absorbance and fluorescence profiles are consistent with a model where the chromophore is formed by a series of reactions with heme or other chromophore precursors and where C34 is essential for binding of the ligand, K92, KI 18 and K130 are involved in transformation into the chromophore and LIPR inhibits the latter reaction. (c) 2006 Elsevier Inc. All rights reserved.