Kulkarni Rohan, Maranholkar Vijay, Nguyen Nam, Cirino Patrick C, Willson Richard C, Varadarajan Navin
Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, United States.
Department of Biology and Biochemistry, University of Houston, Houston, TX, United States.
Front Microbiol. 2024 Mar 8;15:1345617. doi: 10.3389/fmicb.2024.1345617. eCollection 2024.
Agonists of the stimulator of interferon genes (STING) pathway are being explored as potential immunotherapeutics for the treatment of cancer and as vaccine adjuvants for infectious diseases. Although chemical synthesis of 2'3' - cyclic Guanosine Monophosphate-Adenosine Monophosphate (cGAMP) is commercially feasible, the process results in low yields and utilizes organic solvents. To pursue an efficient and environmentally friendly process for the production of cGAMP, we focused on the recombinant production of cGAMP via a whole-cell biocatalysis platform utilizing the murine cyclic Guanosine monophosphate-Adenosine monophosphate synthase (mcGAS). In BL21(DE3) cells, recombinant expression of mcGAS, a DNA-dependent enzyme, led to the secretion of cGAMP to the supernatants. By evaluating the: (1) media composition, (2) supplementation of divalent cations, (3) temperature of protein expression, and (4) amino acid substitutions pertaining to DNA binding; we showed that the maximum yield of cGAMP in the supernatants was improved by 30% from 146 mg/L to 186 ± 7 mg/mL under optimized conditions. To simplify the downstream processing, we developed and validated a single-step purification process for cGAMP using anion exchange chromatography. The method does not require protein affinity chromatography and it achieved a yield of 60 ± 2 mg/L cGAMP, with <20 EU/mL (<0.3 EU/μg) of endotoxin. Unlike chemical synthesis, our method provides a route for the recombinant production of cGAMP without the need for organic solvents and supports the goal of moving toward shorter, more sustainable, and more environmentally friendly processes.
干扰素基因刺激物（STING）通路激动剂正被探索作为治疗癌症的潜在免疫疗法以及传染病疫苗佐剂。尽管2'3'-环磷酸鸟苷-磷酸腺苷（cGAMP）的化学合成在商业上可行，但该过程产率低且使用有机溶剂。为了寻求一种高效且环保的cGAMP生产工艺，我们专注于通过利用小鼠环磷酸鸟苷-磷酸腺苷合酶（mcGAS）的全细胞生物催化平台进行cGAMP的重组生产。在BL21(DE3)细胞中，DNA依赖性酶mcGAS的重组表达导致cGAMP分泌到上清液中。通过评估：（1）培养基组成，（2）二价阳离子的补充，（3）蛋白质表达温度，以及（4）与DNA结合相关的氨基酸取代；我们表明，在优化条件下，上清液中cGAMP的最大产量从146mg/L提高了30%，达到186±7mg/mL。为了简化下游加工，我们开发并验证了一种使用阴离子交换色谱法对cGAMP进行单步纯化的工艺。该方法不需要蛋白质亲和色谱，并且实现了60±2mg/L cGAMP的产量，内毒素含量<20 EU/mL（<0.3 EU/μg）。与化学合成不同，我们的方法提供了一条无需有机溶剂即可重组生产cGAMP的途径，并支持朝着更短、更可持续和更环保工艺发展的目标。
