Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase
Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase
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DOI：
10.1016/j.ymben.2008.04.001
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发表时间：
2008-05-01
影响因子：
8.4
通讯作者：
Swartz, James R.
中科院分区：
文献类型：
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作者：
Goerke, Aaron R.;Loening, Andreas M.;Swartz, James R.
由于其小的尺寸和强烈的发光信号，高斯虫荧光素酶（GLuc）作为一种潜在的成像剂在细胞培养和小动物研究模型中是有吸引力的。然而，使用体内技术的重组GLuc生产仅产生少量活性荧光素酶，可能是由于完全活性需要五个二硫键。无细胞生物学提供了在生物反应中控制催化剂和化学成分的自由，我们利用这一点在无细胞反应中产生大量高活性的GLuc。通过突变细胞提取物来源菌株以减少蛋白质水解、调节反应条件以增强氧化蛋白质折叠、进一步激活能量代谢和促进翻译后激活来提高活性产率。该无细胞蛋白质合成程序产生412 mg/mL纯化GLuc，相对于5 mg/mL分离用于细胞内大肠杆菌表达。无细胞产物的比活性为4.2 × 10（24）光子/s/mol，是任何表征的荧光素酶的最高报告活性。(C)2008年爱思唯尔公司All rights reserved.
Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412 mg/mL of purified GLuc, relative to 5 mg/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2 x 10(24) photons/s/mol, the highest reported activity for any characterized luciferase. (C) 2008 Elsevier Inc. All rights reserved.