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Synthesis of proteins by automated flow chemistry
Open accessPublished version of a preprint
Abstract
Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.
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“However, notorious challenges, including base-induced racemization of seco-acids containing an α-chirality center,47Z/E isomerization of α, β-unsaturated seco-acids,48 and a competitive reaction involving intermolecular esterification leading to the formation of diolide side products have rendered it a formidable task.”
From Ynamide Coupling Reagents: Origin and Advances (2024) · cites this paper as [48]
Citations
Cites
Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide
Merrifield, R. B. (1963) · Journal of the American Chemical Society · doi:10.1021/ja00897a025 · Cited by 7,825
Principles that Govern the Folding of Protein Chains
Anfinsen, C. B. (1973) · Science · doi:10.1126/science.181.4096.223 · Cited by 7,096
Synthesis of Proteins by Native Chemical Ligation
Dawson, P. E., Muir, T. W., Clark‐Lewis, I., & Kent, S. B. H. (1994) · Science · doi:10.1126/science.7973629 · Cited by 4,190
Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53
Oliner, J. D., Pietenpol, J. A., Thiagalingam, S., Gyuris, J., Kinzler, K. W., & Vogelstein, B. (1993) · Nature · doi:10.1038/362857a0 · Cited by 1,474
Lutz, J., Ouchi, M., Liu, D. R., & Sawamoto, M. (2013) · Science · doi:10.1126/science.1238149 · Cited by 1,419
Peptide Coupling Reagents, More than a Letter Soup
El-Faham, A., & Albericio, F. (2011) · Chemical Reviews · doi:10.1021/cr100048w · Cited by 1,245
Synthesis of Native Proteins by Chemical Ligation
Dawson, P. E., & Kent, S. B. H. (2000) · Annual Review of Biochemistry · doi:10.1146/annurev.biochem.69.1.923 · Cited by 1,093
In situ neutralization in Boc‐chemistry solid phase peptide synthesis
Schnölzer, M., Alewood, P. F., Jones, A., Alewood, D., & Kent, S. B. H. (1992) · International journal of peptide & protein research · doi:10.1111/j.1399-3011.1992.tb00291.x · Cited by 1,055
Total chemical synthesis of proteins
Kent, S. B. H. (2008) · Chemical Society Reviews · doi:10.1039/b700141j · Cited by 927
Expanding and reprogramming the genetic code
Chin, J. W. (2017) · Nature · doi:10.1038/nature24031 · Cited by 740
Continuous flow multi-step organic synthesis
Webb, D., & Jamison, T. F. (2010) · Chemical Science · doi:10.1039/c0sc00381f · Cited by 672
Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations
Agouridas, V., Mahdi, O. E., Diemer, V., Cargoët, M., Monbaliu, J. M., & Melnyk, O. (2019) · Chemical Reviews · doi:10.1021/acs.chemrev.8b00712 · Cited by 656
Hydration structure of a collagen peptide
Bella, J., Brodsky, B., & Berman, H. M. (1995) · Structure · doi:10.1016/s0969-2126(01)00224-6 · Cited by 652
Ton-That, H., Liu, G., Mazmanian, S. K., Faull, K. F., & Schneewind, O. (1999) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.96.22.12424 · Cited by 567
Multi-step continuous-flow synthesis
Britton, J., & Raston, C. L. (2017) · Chemical Society Reviews · doi:10.1039/c6cs00830e · Cited by 555
HIV-1 protease: mechanism and drug discovery
Brik, A., & Wong, C. (2002) · Organic & Biomolecular Chemistry · doi:10.1039/b208248a · Cited by 456
Serrano, L. G., Kellis, J. T., Cann, P., Matouschek, A. T., & Fersht, A. R. (1992) · Journal of Molecular Biology · doi:10.1016/0022-2836(92)90562-x · Cited by 382
Buckle, A. M., Schreiber, G., & Fersht, A. R. (1994) · Biochemistry · doi:10.1021/bi00196a004 · Cited by 363
Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins
Bondalapati, S., Jbara, M., & Brik, A. (2016) · Nature Chemistry · doi:10.1038/nchem.2476 · Cited by 359
Energetics of complementary side chain packing in a protein hydrophobic core
Kellis, J. T., Nyberg, K., & Fersht, A. R. (1989) · Biochemistry · doi:10.1021/bi00437a058 · Cited by 348
A fully automated flow-based approach for accelerated peptide synthesis
Mijalis, A. J., Thomas, D. A., Simon, M. D., Adamo, A., Beaumont, R., Jensen, K. F., & Pentelute, B. L. (2017) · Nature Chemical Biology · doi:10.1038/nchembio.2318 · Cited by 330
Self-citationTinkering with Enzymes: What Are We Learning?
Knowles, J. R. (1987) · Science · doi:10.1126/science.3296192 · Cited by 306
Bresson, D. (2006) · Journal of Clinical Investigation · doi:10.1172/jci27191 · Cited by 297
Enzyme replacement and enhancement therapies: lessons from lysosomal disorders
Desnick, R. J., & Schuchman, E. H. (2002) · Nature Reviews Genetics · doi:10.1038/nrg963 · Cited by 268
Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease
Schneider, J., & Kent, S. B. H. (1988) · Cell · doi:10.1016/0092-8674(88)90199-7 · Cited by 234
Barnase and barstar: two small proteins to fold and fit together
Hartley, R. W. (1989) · Trends in Biochemical Sciences · doi:10.1016/0968-0004(89)90104-7 · Cited by 223
Suree, N., Liew, C. K., Villareal, V. A., Thieu, W., Fadeev, E. A., Clemens, J. J., Jung, M. E., & Clubb, R. (2009) · Journal of Biological Chemistry · doi:10.1074/jbc.m109.022624 · Cited by 216
A simple, continuous fluorometric assay for HIV protease
Toth, M. V., & Marshall, G. R. (1990) · International journal of peptide & protein research · doi:10.1111/j.1399-3011.1990.tb00994.x · Cited by 197
Hypersensitive substrate for ribonucleases
Kelemen, B. R., Klink, T. A., Behike, M. A., Eubanks, S. R., Leland, P. A., & Raines, R. T. (1999) · Nucleic Acids Research · doi:10.1093/nar/27.18.3696 · Cited by 194
Palasek, S. A., Cox, Z. J., & Collins, J. M. (2006) · Journal of Peptide Science · doi:10.1002/psc.804 · Cited by 192
Domain size distributions can predict domain boundaries
Wheelan, S. J., Marchler-Bauer, A., & Bryant, S. H. (2000) · Bioinformatics · doi:10.1093/bioinformatics/16.7.613 · Cited by 182 · cited 4×
“Both spectra were obtained by summation of the entire liquid chromatography peak; A) HIV-1 protease (99 amino acids), gradient for analytical HPLC: 5−65% B; B) MDM2[1-118] with His-Tag (127 amino acids) gradient for analytical HPLC: 5−65% B, C) Lysozyme (129 amino acids) gradient for analytical HPLC: 5−65% B, D) FGF1…”
BackgroundMethodInfluentialRapid Flow-Based Peptide Synthesis
Heider, P. L., Adamo, A., Vinogradov, A. A., Li, X., Berger, T., Policarpo, R. L., Zhang, C., Zou, Y., Liao, X., Spokoyny, A. M., et al. (2013) · DSpace@MIT (Massachusetts Institute of Technology) · Cited by 173
Self-citationManufacturing Peptides as Active Pharmaceutical Ingredients
Zompra, A. A., Galanis, A., Werbitzky, O., & Alberício, F. (2009) · Future Medicinal Chemistry · doi:10.4155/fmc.09.23 · Cited by 170
Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme
Durek, T., Torbeev, V. Y., & Kent, S. B. H. (2007) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0610630104 · Cited by 166
Inhibition of the p53-MDM2 Interaction: Targeting a Protein-Protein Interface
Chêne, P. (2004) · Molecular Cancer Research · doi:10.1158/1541-7786.20.2.1 · Cited by 153
Fuse, S., Mifune, Y., & Takahashi, T. (2013) · Angewandte Chemie International Edition · doi:10.1002/anie.201307987 · Cited by 151
Inhibition of the p53-MDM2 interaction: targeting a protein-protein interface.
Chêne, P. (2004) · PubMed · Cited by 136
Loewenthal, R., Sancho, J., & Fersht, A. R. (1991) · Biochemistry · doi:10.1021/bi00241a021 · Cited by 128
Nicholson, E. M., & Scholtz, J. M. (1996) · Biochemistry · doi:10.1021/bi960863y · Cited by 113
The aspartimide problem in Fmoc‐based SPPS. Part I
Mergler, M., Dick, F., Sax, B., Weiler, P., & Vorherr, T. E. (2003) · Journal of Peptide Science · doi:10.1002/psc.430 · Cited by 112
FGF1 — a new weapon to control type 2 diabetes mellitus
Gasser, E., Moutos, C. P., Downes, M., & Evans, R. M. (2017) · Nature Reviews Endocrinology · doi:10.1038/nrendo.2017.78 · Cited by 107
The Folding Mechanism of Barstar: Evidence for Multiple Pathways and Multiple Intermediates
Shastry, M. C. R., & Udgaonkar, J. B. (1995) · Journal of Molecular Biology · doi:10.1006/jmbi.1994.0196 · Cited by 103
High affinity interaction of the p53 peptide-analogue with human Mdm2 and Mdmx
Czarna, A., Popowicz, G. M., Pęcak, A., Wolf, S., Dubin, G., & Holak, T. A. (2009) · Cell Cycle · doi:10.4161/cc.8.8.8185 · Cited by 99
Modular Total Chemical Synthesis of a Human Immunodeficiency Virus Type 1 Protease
Johnson, E. C. B., Malito, E., Shen, Y., RICH, D. H., Tang, W., & Kent, S. B. H. (2007) · Journal of the American Chemical Society · doi:10.1021/ja072870n · Cited by 89
Enhancing Protein Stability with Genetically Encoded Noncanonical Amino Acids
Li, J. C., Liu, T., Wang, Y., Mehta, A. P., & Schultz, P. G. (2018) · Journal of the American Chemical Society · doi:10.1021/jacs.8b07157 · Cited by 88
In-solution enrichment identifies peptide inhibitors of protein–protein interactions
Touti, F., Gates, Z. P., Bandyopadhyay, A., Lautrette, G., & Pentelute, B. L. (2019) · Nature Chemical Biology · doi:10.1038/s41589-019-0245-2 · Cited by 82
Self-citationExploring the potential impact of an expanded genetic code on protein function
Xiao, H., Nasertorabi, F., Choi, S., Han, G. W., Reed, S. A., Stevens, R. C., & Schultz, P. G. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1507741112 · Cited by 79
Solution Structure of Proinsulin
Yang, Y., Hua, Q. X., Liu, J., Shimizu, E., Choquette, M. H., Mackin, R. B., & Weiss, M. A. (2010) · Journal of Biological Chemistry · doi:10.1074/jbc.c109.084921 · Cited by 78
Acid-Mediated Prevention of Aspartimide Formation in Solid Phase Peptide Synthesis
Michels, T., Dölling, R., Haberkorn, U., & Mier, W. (2012) · Organic Letters · doi:10.1021/ol3007925 · Cited by 78
The aspartimide problem in Fmoc-based SPPS. Part III
Mergler, M., & Dick, F. (2005) · Journal of Peptide Science · doi:10.1002/psc.668 · Cited by 78
Protein Thioester Synthesis Enabled by Sortase
Ling, J. J., Policarpo, R. L., Rabideau, A. E., Liao, X., & Pentelute, B. L. (2012) · Journal of the American Chemical Society · doi:10.1021/ja302354v · Cited by 73
Self-citationFlow‐Based Enzymatic Ligation by Sortase A
Policarpo, R. L., Kang, H., Liao, X., Rabideau, A. E., Simon, M. D., & Pentelute, B. L. (2014) · Angewandte Chemie International Edition · doi:10.1002/anie.201403582 · Cited by 72
Self-citationSolid-phase peptide synthesis under continuous-flow conditions.
Lukas, T. J., Prystowsky, M. B., & Erickson, B. W. (1981) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.78.5.2791 · Cited by 67
Protein Prosthesis: A Nonnatural Residue Accelerates Folding and Increases Stability
Arnold, U., Hinderaker, M. P., Köditz, J., Golbik, R. P., Ulbrich‐Hofmann, R., & Raines, R. T. (2003) · Journal of the American Chemical Society · doi:10.1021/ja0351239 · Cited by 66
Gordon, C. P. (2017) · Organic & Biomolecular Chemistry · doi:10.1039/c7ob02759a · Cited by 65
Real-time monitoring of solid-phase peptide synthesis using a variable bed flow reactor
Sletten, E. T., Nuño, M., Guthrie, D., & Seeberger, P. H. (2019) · Chemical Communications · doi:10.1039/c9cc08421e · Cited by 61
Peptide Synthesis Utilizing Micro‐flow Technology
Fuse, S., Otake, Y., & Nakamura, H. (2018) · Chemistry - An Asian Journal · doi:10.1002/asia.201801488 · Cited by 52
Zhan, C., Varney, K. M., Yuan, W., Zhao, L., & Lu, W. (2012) · Journal of the American Chemical Society · doi:10.1021/ja301255n · Cited by 50
Rapid Total Synthesis of DARPin pE59 and Barnase
Mong, S. K., Vinogradov, A. A., Simon, M. D., & Pentelute, B. L. (2014) · ChemBioChem · doi:10.1002/cbic.201300797 · Cited by 45
Self-citationA new support for polypeptide synthesis in columns
Bayer, E., Jung, G., Halász, I., & Sebestian, I. (1970) · Tetrahedron Letters · doi:10.1016/s0040-4039(01)83961-7 · Cited by 42
Recognition of RNase Sa by the Inhibitor Barstar: Structure of the Complex at 1.7 Å Resolution
Sevcík, J., Urbanikova, L., Dauter, Z., & Wilson, K. S. (1998) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444998004429 · Cited by 38
Fluorogenic Assay for Inhibitors of HIV-1 Protease with Sub-picomolar Affinity
Windsor, I. W., & Raines, R. T. (2015) · Scientific Reports · doi:10.1038/srep11286 · Cited by 36
Total synthesis and biochemical characterization of mirror image barnase
Vinogradov, A. A., Evans, E. D., & Pentelute, B. L. (2015) · Chemical Science · doi:10.1039/c4sc03877k · Cited by 35
Self-citationAutomated Flow Synthesis of Tumor Neoantigen Peptides for Personalized Immunotherapy
Truex, N. L., Holden, R. L., Wang, B., Chen, P., Hanna, S., Hu, Z., Shetty, K. S., Olive, O., Neuberg, D. S., Hacohen, N., et al. (2020) · Scientific Reports · doi:10.1038/s41598-019-56943-5 · Cited by 35
Self-citationMutation Choice to Eliminate Buried Free Cysteines in Protein Therapeutics
Xia, X., Longo, L. M., & Blaber, M. (2014) · Journal of Pharmaceutical Sciences · doi:10.1002/jps.24188 · Cited by 26
Total chemical synthesis of human proinsulin
Luisier, S., Avital‐Shmilovici, M., Weiss, M. A., & Kent, S. B. H. (2010) · Chemical Communications · doi:10.1039/c0cc03141k · Cited by 24
Spare, L. K., Laude, V., Harman, D. G., Aldrich-Wright, J. R., & Gordon, C. P. (2018) · Reaction Chemistry & Engineering · doi:10.1039/c8re00190a · Cited by 22
An Improved System for Ribonuclease Ba Expression
Okorokov, A. L., Hartley, R. W., & Panov, K. I. (1994) · Protein Expression and Purification · doi:10.1006/prep.1994.1075 · Cited by 21
Peptide Bond Formations through Flow Chemistry
Ahmed, N. (2017) · Chemical Biology & Drug Design · doi:10.1111/cbdd.13115 · Cited by 21
Editorial overview: Synthetic Biomolecules
Kent, S. B. H., & Alewood, P. F. (2014) · Current Opinion in Chemical Biology · doi:10.1016/j.cbpa.2014.09.037 · Cited by 8
12 Pushing the Limits of Solid-Phase Peptide Synthesis with Continuous Flow
Mijalis, A. J., Steinauer, A., Schepartz, A., & Pentelute, B. L. (2018) · Haug Verlag in Georg Thieme Verlag KG eBooks · doi:10.1055/sos-sd-228-00230 · Cited by 3
Self-citation
Research works printed but not linked: 2
- M. D. Simon “Fast flow biopolymer synthesis ” thesis Massachusetts Institute of Technology Cambridge MA (2017).
- 10.1002/cbic.201300796 · DOI · Paper page
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Metadata
- Authors
- Hartrampf, N., Saebi, A., Poskus, M., Gates, Z. P., Callahan, A. J., Cowfer, A. E., Hanna, S., Antilla, S., Schissel, C. K., Quartararo, A. J., Ye, X., Mijalis, A. J., Simon, M. D., Loas, A., Liu, S., Jessen, C., Nielsen, T. E., & Pentelute, B. L.
- Affiliations
- Massachusetts Institute of Technology (US)
- Novo Nordisk (Denmark) (DK)
- Corresponding author
- Saebi, A.
- Poskus, M.
- Pentelute, B. L.
- ORCID
- Venue
- Science
- Published
- 2020-05-29
- Volume
- 368
- Issue
- 6494
- Pages
- 980-987
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- OpenAlex
- W3029756541
- Cited by
- 367
- References
- 72
- Citations by year
- 2026: 35 · 2025: 61 · 2024: 76 · 2023: 63 · 2022: 44 · 2021: 61 · 2020: 25
- FWCI
- 15.65
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- cc-by-nc-nd
- Locations
- Science (publishedVersion)
- ChemRxiv (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- Royal College of Surgeons in Ireland (submittedVersion, cc-by-nc-nd)
- Funders
- National Science Foundation (1122374)
- Novo Nordisk
- Topics
- Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
- Physical Sciences › Chemical Engineering › Fluid Flow and Transfer Processes › Innovative Microfluidic and Catalytic Techniques Innovation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- protein synthesis, translation elongation, single-domain proteins, protein folding, enzyme activity, protein purification
- MeSH terms
- Protein Domains, Peptides, Proteins, Protein Folding, Solid-Phase Synthesis Techniques
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