System for the expression of recombinant hemoproteins in Escherichia coli
System for the expression of recombinant hemoproteins in Escherichia coli
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DOI：
10.1016/j.pep.2003.12.001
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发表时间：
2004-05-01
影响因子：
1.6
通讯作者：
Goodwin, DC
中科院分区：
文献类型：
--
作者：
Varnado, CL;Goodwin, DC
重组血红素蛋白在大肠杆菌中的表达通常是有限的，因为产生的蛋白质的绝大多数缺乏功能所必需的血红素。这是复杂的事实，大肠杆菌的标准实验室菌株具有有限的能力，从细胞外环境中提取血红素。我们正在开发一种新的工具，旨在增加我们感兴趣的蛋白质的血红素含量，通过简单地补充表达培养基与低浓度的氯化血红素。该血红素蛋白表达（HPEX）系统基于编码来自大肠杆菌的外膜结合血红素受体（ChuA）的质粒（pHPEX 1-pHPEX 3）。coli O157：H7。这种血红素受体和其他类似的受体赋予宿主更有效地内化外源血红素的能力。标准实验室E.大肠杆菌蛋白表达菌株BL-21 [DE 3]与pHPEX质粒的融合表达，导致了一种新蛋白的表达，该蛋白具有适当的分子量为ChuA。该受体的功能，如证明的能力，生长在缺铁培养基补充氯化血红素，能力的未修饰的表达菌株缺乏的。我们感兴趣的蛋白质，过氧化氢酶-过氧化物酶的表达，使用该系统导致血红素含量和活性的显著和平行增加。在前血红素的基础上，HPEX衍生的过氧化氢酶-过氧化物酶的光谱和动力学性质与标准E.基于系统的我们认为pHPEX质粒可能是对其它大肠杆菌的有益补充。大肠杆菌表达系统，并可能有助于解决血红素蛋白结构和功能的广泛问题。(C)2004爱思唯尔公司All rights reserved.
Expression of recombinant hemoproteins in Escherichia coli is often limited because a vast majority of the protein produced lacks the heme necessary for function. This is compounded by the fact that standard laboratory strains of E coli have a limited capacity to withdraw heme from the extracellular environment. We are developing a new tool designed to increase the heme content of our proteins of interest by simply supplementing the expression medium with low concentrations of hemin. This hemoprotein expression (HPEX) system is based on plasmids (pHPEX1-pHPEX3) that encode an outermembrane-bound heme receptor (ChuA) from E. coli O157:H7. This heme receptor, and others like it, confers on the host the ability to more effectively internalize exogenous heme. Transformation of a standard laboratory E. coli protein expression strain (BL-21 [DE3]) with the pHPEX plasmid led to the expression of a new protein with the appropriate molecular weight for ChuA. The receptor was functional as demonstrated by the ability of the transformant to grow on iron-deficient media supplemented with hemin, an ability that the unmodified expression strain lacked. Expression of our proteins of interest, catalase-peroxidases, using this system led to a dramatic and parallel increase in heme content and activity. On a per-heme basis, the spectral and kinetic properties of HPEX-derived catalase-peroxidase were the same as those observed for catalase-peroxidases expressed in standard E. coli-based systems. We suggest that the pHPEX plasmids may be a useful addition to other E. coli expression systems and may help address a broad range of problems in hemoprotein structure and function. (C) 2004 Elsevier Inc. All rights reserved.