High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.
High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.
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DOI：
10.1371/journal.pone.0015491
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发表时间：
2010-11-24
期刊：
影响因子：
3.7
通讯作者：
Swartz JR
中科院分区：
文献类型：
--
作者：
Kuchenreuther JM;Grady-Smith CS;Bingham AS;George SJ;Cramer SP;Swartz JR
用于可再生H2生产的基于氢化酶的技术的实现目前受到对可扩展且高产的方法的需求的限制，以提供活性氢化酶及其所需的成熟酶。在这份报告中，我们描述了一种改进的大肠杆菌为基础的表达系统，能够产生8-30毫克的纯化，活性[FeFe]氢化酶每升的文化，体积产量至少比以前报道的10倍。具体而言，我们克服了与其他体内生产方法相关的两个问题：蛋白质产量低和氢化酶成熟无效。向生长培养基中添加葡萄糖增强了氢化酶表达期间的厌氧代谢和生长，这显著增加了总产率。此外，我们将联合收割机铁和半胱氨酸补充剂与E.大肠杆菌菌株上调铁硫簇蛋白积累。这些措施显着改善体内氢化酶的活化。两个氢化酶，HydA 1从莱茵衣藻和HydA（CpI）从巴氏梭菌，产生与此改进的系统，随后纯化。这些酶的生物物理特性和FTIR光谱分析表明，他们窝藏的H-簇和催化H2进化速率可比的酶从各自的原生生物体分离。我们描述的生产系统将促进基本氢化酶的调查，以及利用这些多产氢酶的新技术的发展。这些方法也可以扩展到生产和研究各种氧敏感的铁硫蛋白以及其他需要缺氧环境的蛋白质。
The realization of hydrogenase-based technologies for renewable H2 production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. In this report, we describe an improved Escherichia coli-based expression system capable of producing 8–30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H2 evolution with rates comparable to those of enzymes isolated from their respective native organisms. The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H2-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.
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影响因子：
3.5
作者：
Czech, Ilka;Silakov, Alexey;Happe, Thomas
通讯作者：
Happe, Thomas
影响因子：
16.6
作者：
Driesener, Rebecca C.;Challand, Martin R.;Roach, Peter L.
通讯作者：
Roach, Peter L.
影响因子：
5
作者：
Akhtar, M. Kalim;Jones, Patrik R.
通讯作者：
Jones, Patrik R.
影响因子：
15
作者：
Hambourger, Michael;Gervaldo, Miguel;Moore, Thomas A.
通讯作者：
Moore, Thomas A.
DOI：
10.1073/pnas.86.13.4932
发表时间：
1989-07-01
影响因子：
11.1
作者：
ADAMS, MWW;ECCLESTON, E;HOWARD, JB
通讯作者：
HOWARD, JB