Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis
Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis
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DOI：
10.1002/bit.20529
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发表时间：
2005-08-20
影响因子：
3.8
通讯作者：
Puglisi, JD
中科院分区：
文献类型：
--
作者：
Underwood, KA;Swartz, JR;Puglisi, JD
随着产量的增加和成本的降低，无细胞蛋白质合成（CFPS）越来越多地用于蛋白质生产。 CFPS 优化工作主要集中在能量供应和小分子代谢上，但人们对蛋白质合成机制或限制蛋白质合成速率的因素知之甚少。在这里，定量多核糖体谱分析用于表征无细胞翻译，从而阐明许多动力学参数。基于大肠杆菌的 CFPS 反应中的核糖体浓度为 1.6 +/- 0.1 muM，其中 72 +/- 4% 以最大蛋白质合成速率进行主动翻译。确定每个核糖体每秒 1.5 +/- 0.2 个氨基酸的翻译延伸率和 8.2 x 10(-9)+/- 0.3 x 10(-9) M/s 的起始速率，这与每个 mRNA 平均每 60 +/- 9 s 一次起始相关。测得的 CFPS 起始和伸长率比体内速率低一个数量级，进一步分析确定伸长是主要限制。在 CFPS 反应中添加纯化的延伸因子 (EF) 增加了核糖体延伸率、蛋白质合成率和产量，以及翻译起始率，表明起始和延伸之间可能存在耦合。对无细胞系统中翻译起始的进一步检查表明，mRNA 的第一次起始比后续起始慢。我们的结果表明，多核糖体分析是表征无细胞翻译和识别限制步骤的有效工具，翻译因子的稀释是 CFPS 的限制，并且 CFPS 是对翻译进行新颖观察的有用平台。 (c) 2005 年 Wiley 期刊公司。
Cell-free protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. CFPS optimization efforts have focused primarily on energy supply and small molecule metabolism, though little is known about the protein synthesis machinery or what limits protein synthesis rates. Here, quantitative polysome profile analysis was used to characterize cell-free translation, thereby elucidating many kinetic parameters. The ribosome concentration in Escherichia coli-based CFPS reactions was 1.6 +/- 0.1 mu M, with 72 +/- 4% actively translating at maximal protein synthesis rate. A translation elongation rate of 1.5 +/- 0.2 amino acids per second per ribosome and an initiation rate of 8.2 x 10(-9)+/- 0.3 x 10(-9) M/s, which correlates to, on average, one initiation every 60 +/- 9 s per mRNA, were determined. The measured CFPS initiation and elongation rates are an order of magnitude lower than the in vivo rates and further analysis identified elongation as the major limitation. Adding purified elongation factors (EFs) to CFPS reactions increased the ribosome elongation rate and protein synthesis rates and yields, as well as the translation initiation rate, indicating a possible coupling between initiation and elongation. Further examination of translation initiation in the cell-free system showed that the first initiation on an mRNA is slower than subsequent initiations. Our results demonstrate that polysome analysis is a valid tool to characterize cell-free translation and to identify limiting steps, that dilution of translation factors is a limitation of CFPS, and that CFPS is a useful platform for making novel observations about translation. (c) 2005 Wiley Periodicals, Inc.