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一种细胞毒性癌症治疗药物的无细胞蛋白质合成:核糖核酸酶Onconase的生产及一种即加水无细胞系统

Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system.

作者信息

Salehi Amin S M, Smith Mark Thomas, Bennett Anthony M, Williams Jacob B, Pitt William G, Bundy Bradley C

机构信息

Department of Chemical Engineering, Brigham Young University, Provo, Utah, USA.

出版信息

Biotechnol J. 2016 Feb;11(2):274-81. doi: 10.1002/biot.201500237. Epub 2015 Nov 25.

Abstract

Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult-to-express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell-free protein synthesis. Cell-free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these cell-free systems make them compelling candidates for future biotherapeutic screening and production.

摘要

生物疗法有许多有前景的应用,如抗癌治疗、免疫抑制和疫苗。然而,由于它们的生物学性质,一些生物疗法通过传统重组方法快速表达和筛选活性可能具有挑战性。例如,难以表达的蛋白质可能具有细胞毒性,或者在表达过程中形成包涵体,增加了纯化和下游表征的时间、工作量和难度。简化此类疗法表达和筛选的一个潜在途径是利用无细胞蛋白质合成。无细胞系统由于其开放且可塑的反应环境,为体内生产提供了一个有吸引力的替代方案。在这项工作中,我们展示了使用无细胞系统表达和直接筛选难以表达的细胞毒性蛋白核糖核酸酶。使用无细胞系统,核糖核酸酶可以快速以可溶的活性形式表达。此外,反应环境的开放性允许直接和即时的下游表征,而无需纯化。此外,我们报告了一种“即加水”冻干无细胞系统生产核糖核酸酶的能力。这种冻干系统在高于冰点的温度下储存长达一年后仍保持活力。这些无细胞系统的有益特性使其成为未来生物疗法筛选和生产的有力候选者。

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