The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli
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 comm...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2024-03-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1345617/full |
| _version_ | 1797269033830055936 |
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| author | Rohan Kulkarni Vijay Maranholkar Nam Nguyen Patrick C. Cirino Richard C. Willson Navin Varadarajan |
| author_facet | Rohan Kulkarni Vijay Maranholkar Nam Nguyen Patrick C. Cirino Richard C. Willson Navin Varadarajan |
| author_sort | Rohan Kulkarni |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-25T01:41:57Z |
| format | Article |
| id | doaj.art-3ffd210b80c846e1be8aa7892fce9a4c |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-25T01:41:57Z |
| publishDate | 2024-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-3ffd210b80c846e1be8aa7892fce9a4c2024-03-08T04:46:49ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2024-03-011510.3389/fmicb.2024.13456171345617The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coliRohan Kulkarni0Vijay Maranholkar1Nam Nguyen2Patrick C. Cirino3Richard C. Willson4Navin Varadarajan5Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, United StatesDepartment of Biology and Biochemistry, University of Houston, Houston, TX, United StatesDepartment of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, United StatesDepartment of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, United StatesDepartment of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, United StatesDepartment of Chemical and Biomolecular Engineering, University of Houston, Houston, TX, United StatesAgonists 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.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1345617/fullSTING agonists2′3’-cGAMP productionEscherichia coli bacterial expressionlipopolysaccharide removalmurine cGAS enzyme |
| spellingShingle | Rohan Kulkarni Vijay Maranholkar Nam Nguyen Patrick C. Cirino Richard C. Willson Navin Varadarajan The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli Frontiers in Microbiology STING agonists 2′3’-cGAMP production Escherichia coli bacterial expression lipopolysaccharide removal murine cGAS enzyme |
| title | The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli |
| title_full | The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli |
| title_fullStr | The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli |
| title_full_unstemmed | The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli |
| title_short | The efficient synthesis and purification of 2′3’- cGAMP from Escherichia coli |
| title_sort | efficient synthesis and purification of 2 3 cgamp from escherichia coli |
| topic | STING agonists 2′3’-cGAMP production Escherichia coli bacterial expression lipopolysaccharide removal murine cGAS enzyme |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1345617/full |
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