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首页> 外文期刊>Biotechnology Progress >Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis
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Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis

机译：使用无细胞蛋白质合成技术快速表达和纯化100 nmol量的活性蛋白质

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Two strategies for ATP regeneration during cell-free protein synthesis were applied to the large-scale production and single-column purification of active chloramphenicol acetyl transferase (CAT). Fed-batch reactions were performed on a 5-10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT in a 5 mL reaction over the course of 5 h. The PANOx system produced 261 +/- 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatography adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed-batch cell-free protein synthesis reaction.
机译：无细胞蛋白质合成过程中ATP再生的两种策略已应用于大规模生产和活性氯霉素乙酰转移酶（CAT）的单列纯化。补料分批反应以5-10 mL的规模进行，比典型的反应体积大约2个数量级。丙酮酸氧化酶系统在5小时内的5 mL反应中生成104 nmol的活性CAT。 PANOx系统在4小时内以10 mL反应产生了261 +/- 42 nmol（约7 mg）的活性CAT。通过简单的亲和色谱吸附和洗脱将反应产物纯化至表观均匀性，产率约为70％。据我们所知，这是在简单的补料分批无细胞蛋白质合成反应中报道的最大量的主动表达蛋白质。

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