Biological Systems Engineering Department, 210-A Seitz Hall, Virginia Polytechnic Institute and State University, Blacksburg, Virgina 24061, USA.
Microb Cell Fact. 2009 Jun 4;8:30. doi: 10.1186/1475-2859-8-30.
Thermostable enzymes from thermophilic microorganisms are playing more and more important roles in molecular biology R&D and industrial applications. However, over-production of recombinant soluble proteins from thermophilic microorganisms in mesophilic hosts (e.g. E. coli) remains challenging sometimes.
An open reading frame TM0438 from a hyperthermophilic bacterium Thermotoga maritima putatively encoding 6-phosphogluconate dehydrogenase (6PGDH) was cloned and expressed in E. coli. The purified protein was confirmed to have 6PGDH activity with a molecular mass of 53 kDa. The kcat of this enzyme was 325 s-1 and the Km values for 6-phosphogluconate, NADP+, and NAD+ were 11, 10 and 380 muM, respectively, at 80 degrees C. This enzyme had half-life times of 48 and 140 h at 90 and 80 degrees C, respectively. Through numerous approaches including expression vectors, hosts, cultivation conditions, inducers, and codon-optimization of the 6pgdh gene, the soluble 6PGDH expression levels were enhanced to ~250 mg per liter of culture by more than 500-fold. The recombinant 6PGDH accounted for >30% of total E. coli cellular proteins when lactose was used as a low-cost inducer. In addition, this enzyme coupled with glucose-6-phosphate dehydrogenase for the first time was demonstrated to generate two moles of NADPH per mole of glucose-6-phosphate.
We have achieved a more than 500-fold improvement in the expression of soluble T. maritima 6PGDH in E. coli, characterized its basic biochemical properties, and demonstrated its applicability for NADPH regeneration by a new enzyme cocktail. The methodology for over-expression and simple purification of this thermostable protein would be useful for the production of other thermostable proteins in E. coli.
来自嗜热微生物的热稳定酶在分子生物学研发和工业应用中发挥着越来越重要的作用。然而，在嗜温宿主（如大肠杆菌）中过量生产来自嗜热微生物的重组可溶性蛋白有时仍然具有挑战性。
从一种超嗜热细菌 Thermotoga maritima 中克隆并在大肠杆菌中表达了一个开放阅读框 TM0438，该框编码 6-磷酸葡萄糖酸脱氢酶（6PGDH）。纯化的蛋白质被证实具有 6PGDH 活性，分子量为 53 kDa。该酶的 kcat 为 325 s-1，在 80°C 时，6-磷酸葡萄糖酸、NADP+和 NAD+的 Km 值分别为 11、10 和 380 μM。该酶在 90°C 和 80°C 下的半衰期分别为 48 和 140 h。通过包括表达载体、宿主、培养条件、诱导剂和 6pgdh 基因的密码子优化在内的多种方法，将可溶性 6PGDH 的表达水平提高了 500 多倍，达到约 250 mg/L 培养物。当使用乳糖作为低成本诱导剂时，重组 6PGDH 占大肠杆菌总细胞蛋白的比例超过 30%。此外，首次证明该酶与葡萄糖-6-磷酸脱氢酶偶联，可每摩尔葡萄糖-6-磷酸生成 2 摩尔 NADPH。
我们在大肠杆菌中实现了可溶性 T. maritima 6PGDH 的表达提高了 500 多倍，表征了其基本生化特性，并通过新的酶混合物证明了其用于 NADPH 再生的应用。这种过表达和简单纯化这种热稳定蛋白的方法将有助于在大肠杆菌中生产其他热稳定蛋白。
