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从大肠杆菌中有效合成和净化2'3'-cGAMP

Rohan Kulkarni¹, Vijay Maranholkar², Nam Nguyen¹

¹Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, United States.

Frontiers in microbiology

|March 25, 2024

概括

本研究提出了一种可持续的全细胞生物催化方法,用于生产2'3' - 循环诺辛单酸-诺辛单酸 (cGAMP). 大肠杆菌中cGAMP的优化重组生产显著提高了产量,并简化了净化,为化学合成提供了一个环保的替代方案.

科学领域:

生物技术是生物技术.
生物化学生物化学
免疫学免疫学免疫学

背景情况:

干扰素基因刺激器 (STING) 途径激动剂,如2'3' - 循环诺辛单酸-诺辛单酸 (cGAMP),对于癌症免疫疗法和疫苗辅助剂至关重要.
化学合成cGAMP面临挑战,包括低产量和使用有机溶剂,需要更绿色的生产方法.

研究的目的:

使用全细胞生物催化剂开发一个高效和环保的cGAMP复合生产平台.
为了优化表达和纯化的cGAMP产生的鼠类循环氨酸单酸-氨酸单酸合成酶 (mcGAS) 酶在大肠杆菌.

主要方法:

在大肠杆菌BL21(DE3) 中,用于全细胞生物催化剂的小鼠循环氨酸单酸-氨酸单酸合成酶 (mcGAS) 的重组表达.
优化发酵条件,包括介质组成,双价补充和表达温度.
为cGAMP开发单步离子交换色谱净化工艺.

主要成果:

在优化条件下,超水生物的cGAMP产量增加了30%,从146 mg/L增加到186 ± 7 mg/L.
一个简化的,单步离子交换色谱净化得到了60±2 mg/L的cGAMP与低内毒素水平 (<20 EU/mL).
再组合方法避免有机溶剂,为化学合成提供可持续的替代方案.

结论:

使用重组mcGAS的全细胞生物催化剂为cGAMP生产提供了高效和可持续的途径.
优化过程和简化净化简化了cGAMP制造,支持其治疗应用.
这种环保方法符合制药制造中对可持续生物工艺的日益增长的需求.

关键词:

2′3-cGAMP的生产方式.大肠杆菌的细菌表达.刺痛的激动剂是刺痛的激动剂清除脂多糖糖的方法鼠类cGAS酶的使用

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