Point-of-Care Peptide Hormone Production Enabled by Cell-Free Protein Synthesis

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.

A cell-free workflow for detecting and characterizing RiPP recognition element-precursor peptide interactions

“…Encouraged by these results, we then tested the functionality of these RRE-containing proteins in an AlphaLISA based reaction with each of the RREs’ respective peptide substrates. AlphaLISA is an in-solution, bead-based assay version of ELISA that is amenable to acoustic liquid handling robots and small (2 μL) reaction sizes in a 384-well plate format 32 and has previously been used with cell-free systems to assess protein-protein 46,47 and protein-peptide interactions 46,48 .…”

Ribosome Pool Engineering Increases Protein Biosynthesis Yields

“…The donor and acceptor beads were then added to the wells and incubated for an additional 1 h at room temperature. Chemiluminescence was measured on a Tecan Infinite M1000 Pro using the AlphaLISA filter with an excitation time of 100 ms, an integration time of 300 ms, and a settle time of 20 ms after 10 min of incubation inside the instrument as previously reported …”

“…Chemiluminescence was measured on a Tecan Infinite M1000 Pro using the AlphaLISA filter with an excitation time of 100 ms, an integration time of 300 ms, and a settle time of 20 ms after 10 min of incubation inside the instrument as previously reported. 62 iSAT Reactions. iSAT reactions were assembled with four 5-μL replicates per rRNA construct being tested based on previous work.…”

A Cell-Free Protein Synthesis Platform to Produce a Clinically Relevant Allergen Panel

“…Here, we describe a cell-free platform for producing recombinant allergens that overcomes the shortcomings of traditional allergen extract preparations and endows several favorable features beyond the capabilities of cell-based recombinant methods. Cell-free protein synthesis (CFPS) − is a technique that has been applied to protein biologic discovery, , glycoprotein synthesis, − point-of-care manufacturing, − distribution of medicines to resource-limited settings, , small molecules, − diagnostics, − and education. − …”