Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation.
Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation.
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DOI：
10.1186/1475-2859-12-21
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发表时间：
2013-02-27
影响因子：
6.4
通讯作者：
Casal M
中科院分区：
文献类型：
--
作者：
Collins T;Azevedo-Silva J;da Costa A;Branca F;Machado R;Casal M
丝弹性蛋白样蛋白（SELP）结合了丝和弹性蛋白的理化和生物特性，在制药、再生医学和材料领域具有很高的应用潜力。然而，它们的使用开发受到其生产水平的限制。在这里，我们描述了 pET-E 中最近描述的新型 SELP 的批量生产优化。大肠杆菌BL21(DE3)表达系统。采用全面的经验方法检查所有过程变量（介质、诱导时间和周期、温度、pH、通气和搅拌）以及生物过程的详细表征，试图最大限度地提高该系统的产量。本研究表明，使用 pH 6-7.5 的优质肉汤、摇瓶体积与培养基体积比为 10:1、搅拌速度为 200 rpm，在 37°C 下实现最大 SELP 体积产量。在稳定期开始时使用 0.5 mM IPTG 实现最大诱导，诱导期至少为 4 小时。我们表明，选择剂氨苄青霉素和羧苄青霉素在培养早期迅速降解，并且质粒稳定性在诱导时急剧下降。此外，乙酸盐在生物过程中累积至对宿主细胞具有抑制作用的水平。使用我们的优化条件，获得了 500 mg/L 的纯化 SELP。我们已经确定了新型 SELP 的摇瓶生产的最佳条件，最终生产水平是迄今为止报道的最高水平。虽然这项研究的重点是 SELP，但我们相信，任何使用 pET（氨苄青霉素选择性标记）-E 的研究也可能会引起普遍兴趣。使用大肠杆菌BL21(DE3)表达系统。特别是，我们表明诱导时间在该系统中至关重要，与通常认为的相反，通过在稳定期开始时的诱导获得最佳产量。此外，我们相信我们所使用的系统处于或接近最大生产力，质粒编码的β-内酰胺酶快速降解选择剂、诱导时的质粒不稳定性和高乙酸盐产量水平是进一步提高产量的主要限制因素。
Silk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system. This study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6–7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained. We have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.
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影响因子：
6.2
作者：
Qiu, Weiguo;Huang, Yiding;Teng, Weibing;Cohn, Celine M.;Cappello, Joseph;Wu, Xiaoyi
通讯作者：
Wu, Xiaoyi
影响因子：
3.7
作者：
Machado R;Azevedo-Silva J;Correia C;Collins T;Arias FJ;Rodríguez-Cabello JC;Casal M
通讯作者：
Casal M
影响因子：
3.2
作者：
Arnold, CN;McElhanon, J;Siegele, DA
通讯作者：
Siegele, DA
影响因子：
4.8
作者：
Collins, T;Meuwis, MA;Gerday, C
通讯作者：
Gerday, C
影响因子：
2.9
作者：
Chow, Dominic C.;Dreher, Matthew R.;Chilkoti, Ashutosh
通讯作者：
Chilkoti, Ashutosh