| Summary: | Flow chemistry techniques and methods have given the broad scientific community high-fidelity access to chemical compounds with minimal effort compared to traditional synthetic techniques. Since the introduction of solid phase peptide synthesis (SPPS), the peptide community has endeavored to combine the convenience of flow chemistry with the iterative steps associated with peptide elongation in SPPS. Nearly one decade ago, members of the Pentelute lab envisioned and developed a flow-based peptide synthesizer, the Automated Fast-Flow Peptide Synthesizer, or AFPS for short. This technology enabled fast, reliable access to short peptide chains, with each coupling taking less than 3 minutes in total, significantly decreasing the labor needed to produce these peptides. However, peptide chains over 50 amino acids remained challenging to produce via AFPS, microwave synthesis, or traditional SPPS batch couplings. With modern research requiring rapid and high-fidelity access to long polypeptide chains, an immediate need to develop peptide synthesis technology to produce single-domain protein polypeptides in a single shot will be critical. Herein, I report on the arduous journey and unmatched teamwork needed to improve the AFPS systems for regular, reliable access to polypeptide chains of more than 200 amino acids in a single working day. In addition, I will highlight the workflow and knowledge needed to take a free polypeptide chain to a fully folded and biologically active protein, equivalent in form and function to its recombinant counterparts. I will discuss the iterative steps my team took to vary both chemical and mechanical and control variables to improve per-coupling yield enough to enable access to full-length single-domain proteins. On this journey, we utilized test peptides to validate synthesis quality and later synthesized a suite of full-length single-domain biologically active proteins. I will spend some time focusing on the barnase-barstar binding pair. Next, I will dive into how I build and design each AFPS synthesizer to improve synthesis outcomes and user-friendliness while retaining the core functionality and customizability that have made the AFPS so successful in the Pentelute lab. I will highlight my role in the renovation of the first generation AFPS system, the “Automatide,” and dive into the key characteristics that set our synthesizers apart from what is currently commercially available. Finally, we report on the synthesis and characterization of several small and very interesting luciferases. Luciferases are proteins that produce bioluminescence when exposed to specific chemical substrates, and for the organisms that produce these enzymes, they play a vital role in mating, defense, and camouflage. In the research arena, luciferases have had broad applications for decades, including detection of environmental contaminants, diagnosis of pathogens, high-throughput screening for drug discovery, understanding protein-protein interactions, and more. Current efforts in the field have focused on the development of small artificial luciferases due to their many advantages over traditional larger luciferases, such as enhanced stability and increased brightness. Herein, we report on the synthesis and characterization of the copepod, Gaussia priceps, luciferase GLuc (18 kDa), and artificial luciferases picALuc (12 kDa) and LuxSit-I (14 kDa). In addition, we synthesized the mirror-image counterpart of picALuc due to its potential for broad-reaching impact in health and diagnostics; this is the first reported mirror-image bioluminescent luciferase. Finally, we will report on our efforts to develop a split-picAluc protein complement assay (PCA) using AS-MS technology, which will be the smallest and most versatile split-luciferase reported to date. In summary, fast-flow peptide synthesis was utilized to produce and investigate several biologically relevant proteins to improve upon existing tools available to the broad chemistry and biology community.
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