Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
PLoS One. 2012;7(4):e35886. doi: 10.1371/journal.pone.0035886. Epub 2012 Apr 26.
Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies.
We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L(-1) of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg(-1) min(-1)).
The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.
由于复杂的、酶特异性的翻译后成熟过程，金属酶（包括氢化酶）的高效表达和纯化具有挑战性。成熟的保真度低会导致制备物中含有大量无活性的脱辅基蛋白，这些蛋白不适合生物物理或晶体学研究。
我们描述了一种融合蛋白的构建、过表达和高产纯化，该融合蛋白由藻类[2Fe2S]-铁氧还蛋白 PetF（Fd）和[FeFe]-氢化酶 HydA1 组成。通过改进表达时使用的培养条件和培养基成分，优化了 Fd-HydA1 的成熟。我们还证明，与 C 端融合相比，Fd 与 HydA1 的 N 端融合导致表达水平提高了 4 倍。这些改进共同导致了 HydA1 活性的增强和纯化后的产量提高。Fd 与 DEAE 的强结合亲和力允许通过离子交换和 StrepTactin 亲和层析进行两步纯化。此外，在 Fd-HydA1 接头中加入 TEV 蛋白酶位点允许在 DEAE 步骤后通过 StrepTactin 进行蛋白酶切去除 Fd，并单独纯化 HydA1。结合使用，该工艺使大肠杆菌培养物中每升 5 毫克的 HydA1 产量达到 5 毫克 L(-1)，比活为 1000 U（U=1µmol 氢气产生 mg(-1) min(-1)）。
[FeFe]-氢化酶是高效的酶，它们的催化位点为开发稳健的氢活化催化剂提供了合成模型结构。为了更详细地研究其结构-功能特性，并将氢化酶用于生物技术应用，需要可靠的快速、高产表达和纯化方法。
