Abstract
In resource-limited settings, it can be difficult to safely deliver sensitive biologic medicines to patients due to cold chain and infrastructure constraints. Point-of-care drug manufacturing could circumvent these challenges since medicines could be produced locally and used on-demand. Toward this vision, we combine cell-free protein synthesis (CFPS) and a 2-in-1 affinity purification and enzymatic cleavage scheme to develop a platform for point-of-care drug manufacturing. As a model, we use this platform to synthesize a panel of peptide hormones, an important class of medications that can be used to treat a wide variety of diseases including diabetes, osteoporosis, and growth disorders. With this approach, temperature-stable lyophilized CFPS reaction components can be rehydrated with DNA encoding a SUMOylated peptide hormone of interest when needed. Strep-Tactin affinity purification and on-bead SUMO protease cleavage yield peptide hormones in their native form that are recognized by ELISA antibodies and that can bind their respective receptors. With further development to ensure proper biologic activity and patient safety, we envision that this platform could be used to manufacture valuable peptide hormone drugs in a decentralized way.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1216-1226 |
| Number of pages | 11 |
| Journal | ACS synthetic biology |
| Volume | 12 |
| Issue number | 4 |
| DOIs | |
| State | Published - Apr 21 2023 |
Funding
This work was supported by the Defense Threat Reduction Agency (HDTRA1-21-1-0038) and the National Science Foundation (MCB 1936789). A.H.T. was supported by the National Institutes of Health National Research Service Award Fellowship (1F31AI165279). L.G. was supported by the National Science Foundation Graduate Research Fellowship (DGE-2234667). This work made use of the IMSERC MS facility at Northwestern University, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-2025633), and Northwestern University. This work also used the Keck Biophysics Facility, a shared resource of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University supported in part by the NCI Cancer Center Support Grant #P30 CA060553. We acknowledge the support of the High Throughput Analysis Laboratory at Northwestern University. We would like to thank Dr. Benjamin Owen and Dr. Fernando Tobias for training and assistance with mass spectrometry and Dr. Arabela Grigorescu for training and assistance with biolayer interferometry.
Keywords
- affinity purification
- cell-free protein synthesis
- enzymatic cleavage
- peptide hormones
- point-of-care manufacturing
ASJC Scopus subject areas
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)
