细胞工程策略可溶性和分泌性重组人体胰岛素的生产在Pseudomonas光体和批量生物反应器研究研究
Ansuman Sahoo1, Venkata Dasu Veeranki1, Sanjukta Patra2
1Biochemical Engineering Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, 781039, Assam, India.
Journal of biotechnology
|June 13, 2025
概括
这项研究增强了Pseudomonas fluorescens中可溶性人体胰岛素的产生,通过共同表达像DsbA和Pbp这样的陪伴物. 优化条件显著增加了蛋白质产量和溶解度,在生物反应器中达到高位数.
科学领域:
- 生物技术是生物技术.
- 蛋白质表达方式
- 再组合蛋白质的生产生产.
背景情况:
- 再组合人体胰岛素生产面临的挑战包括体的形成.
- 之前的工作在Pseudomonas fluorescens中实现了胰岛素表达,但降低温度降低了蛋白质标位.
- 可溶性蛋白质的表达需要克服聚合的策略.
研究的目的:
- 分析伴侣和信号对可溶性人体胰岛素合成的个人和协同效应.
- 为了优化条件,增强重组蛋白质生产.
- 研究His-tag位置和等离子体起源对表达的影响.
主要方法:
- 通过使用抗分胺素等离子体的等离子体 (脱硫结合蛋白A - DsbA,酸盐结合蛋白 - Pbp) 的联合表达.
- 评估His标签位置对蛋白质表达和可溶性的影响.
- 优化抗生素度,诱导剂水平,注射百分比和诱导光学密度 (OD600).
- 批量生物反应器培养和循环二元化分析用于结构验证.
主要成果:
- 结合DsbA和Pbp的共同表达产生了~60%的可溶性聚变蛋白.
- 一个复制的高副本起源降低了可溶性蛋白质标位,增加了滞后阶段.
- 优化条件使总体蛋白质和可溶蛋白质分数度分别增加了58%和27%.
- 在一个批量生物反应器中,最大达到234.58 mg/l的融合蛋白,溶解率为71.59%.
- 纯化胰岛素通过循环二元化表现出与标准胰岛素类似的二次结构.
结论:
- 特定的伴侣和信号的联合表达显著增强可溶性人体胰岛素的产生.
- 优化表达参数和等离子体元素对于最大限度地提高重组蛋白的产量和可溶性至关重要.
- 这项研究提出了一种新的可溶性胰岛素生产的组合方法,实现了高产量和纯度.
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