Wu Chang-Hao, Ponir Cynthia A, Haja Dominik K, Adams Michael W W
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA.
Protein Eng Des Sel. 2018 Sep 1;31(9):337-344. doi: 10.1093/protein/gzy025.
The NADPH-dependent cytoplasmic [NiFe]-hydrogenase (SHI) from the hyperthermophile Pyrococcus furiosus, which grows optimally near 100°C, is extremely thermostable and has many in vitro applications, including cofactor generation and hydrogen production. In particular, SHI is used in a cell-free synthetic pathway that contains more than a dozen other enzymes and produces three times more hydrogen (12 H2/glucose) from sugars compared to cellular fermentations (4 H2/glucose). We previously reported homologous over-expression and rapid purification of an affinity-tagged (9x-His) version of SHI, which is a heterotetrameric enzyme. However, about 30% of the enzyme that was purified contained an inactive trimeric form of SHI lacking the catalytic [NiFe]-containing subunit. Herein, we constructed a strain of P. furiosus that contained a second set of the eight genes involved in the maturation of the catalytic subunit and insertion of the [NiFe]-site, along with a second set of the four genes encoding the SHI structural subunits. This resulted in a 40% higher yield of the purified affinity-tagged enzyme and the content of the inactive trimeric form decreased to 5% of the total protein. These results bode well for the future production of active SHI for both basic and applied purposes.
嗜热栖热菌（Pyrococcus furiosus）的NADPH依赖性胞质[NiFe]氢化酶（SHI）在接近100°C的环境中生长最佳，具有极高的热稳定性，并在许多体外应用中发挥作用，包括辅因子生成和氢气生产。特别是，SHI被用于一种无细胞合成途径，该途径包含十几种其他酶，与细胞发酵（4 H2/葡萄糖）相比，从糖类中产生的氢气量增加了两倍（12 H2/葡萄糖）。我们之前报道了亲和标签（9x-组氨酸）版本的SHI的同源过表达和快速纯化，SHI是一种异源四聚体酶。然而，纯化得到的约30%的酶含有无活性的三聚体形式的SHI，缺少含催化性[NiFe]的亚基。在此，我们构建了一株嗜热栖热菌，它含有第二套参与催化亚基成熟和[NiFe]位点插入的八个基因，以及第二套编码SHI结构亚基的四个基因。这使得纯化的亲和标签酶产量提高了40%，无活性三聚体形式的含量降至总蛋白的5%。这些结果对于未来为基础和应用目的生产活性SHI来说是个好兆头。
