# The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli

## TL;DR

This paper describes a new, efficient, and eco-friendly method to produce a potential cancer immunotherapy molecule using bacteria instead of chemical synthesis.

## Contribution

A recombinant whole-cell biocatalysis platform for producing cGAMP in E. coli with optimized yield and a single-step purification method.

## Key findings

- Recombinant expression of mcGAS in E. coli increased cGAMP yield by 30% under optimized conditions.
- Anion exchange chromatography enabled single-step purification of cGAMP with high yield and low endotoxin.
- The method avoids organic solvents and supports sustainable production of cGAMP.

## Abstract

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 E. coli 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.