Peptide synthesis was outsourced to CPC Scientific.
Abstract
Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.
SOCIAL MEDIA
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Lin, Wilson, Eduardo Aluicio-Sarduy, Hailey A. Houson, Todd E. Barnhart, Volkan Tekin, Justin J. Jeffery, Ashley M. Weichmann, Kendall E. Barrett, Suzanne E. Lapi, and Jonathan W. Engle. Nuclear Medicine and Biology) 118 (2023): 108329.
Lyophilized NOTA-NT-20.3 (Ac-Lys(NOTA)-Pro-NMeArg-Arg-Pro-Tyr-Tle-Leu, 1383.6 g/mol, “NT”, CPC Scientific) was diluted to 1 mg/mL in ultrapure water (Milli-Q, >18.2 MΩ-cm) and used without further purification.
Development of a selective, sensitive and robust oxyntomodulin dual monoclonal antibody immunoassay.
Umberger, T.S., Ming, W., Cox, J.M., Konrad, R.J. and Siegel, R.W. Bioanalysis 14, no. 18 (2022): 1229-1239.
- Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, IN46285, USA
Human K2 EDTA and P800 plasma (500 μl) was spiked with proglucagon 33–61, 35–61 and 36–61 stable-isotope-labeled internal standard peptides (CPC Scientific, custom order) and diluted with I buffer (25 mmol/l Tris-HCl, 25 mmol/l HEPES, 300 mmol/l NaCl, 0.1% (v/v) octyl β-D-glucopyranoside, pH 7.5).
Line, J.E.; Seal, B.S.; Garrish, J.K. Appl. Microbiol. 2022, 2, 688–700.
Peptides were synthesized using standard solid-phase(Fmoc) chemistry with a peptide synthesizer (CPC Scientific Inc., Sunnyvale, CA 94089,USA, C12K-2β12 [..]
Kirk, N.S., Chen, Q., Wu, Y.G., Asante, A.L., Hu, H., Espinosa, J.F., Martínez-Olid, F., Margetts, M.B., Mohammed, F.A., Kiselyov, V.V. and Barrett, D.G. Nature Communications 13, no. 1 (2022): 5695.
- Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.
Peptides were synthesized under contract by CPC Scientific, except for the N-terminally acetylated version of IM172N22 and the Glu3Arg, Glu3Ala, Glu4Arg, Glu4Ala, Glu5Ala, Glu5Arg, Trp6Ala, Gln8Ala, Ile9Ala, Glu10Ala, Glu10Arg and Tyr14Ala mutants of IM172N22
Coskun, T., Urva, S., Roell, W.C., Qu, H., Loghin, C., Moyers, J.S., O’Farrell, L.S., Briere, D.A., Sloop, K.W., Thomas, M.K. and Pirro, V. Cell Metabolism 34, no. 9 (2022): 1234-1247.
- Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Homologous and heterologous competition experiments were performed with non-radioactive peptide analogues[127I]-Tyr1-GIP(1-42) and [127]-Tyr10-GIP(1-42) to ensure quantification of the high-affinity binding site of the GIPR. Peptide analogues were generated using synthetic [127I]-Tyr amino acid building blocks (CPC Scientific).
Cecil, D.L., Curtis, B., Gad, E., Gormley, M., Timms, A.E., Corulli, L., Bos, R., Damle, R.N., Sepulveda, M.A. and Disis, M.L. Scientific Reports 12, no. 1 (2022): 13618.
- Cancer Vaccine Institute, University of Washington, 850 Republican Street, Brotman Bld., 2nd Floor, Box 358050, Seattle, WA 98195-8050, USA.
- Janssen Research and Development LLC, Spring House, PA, USA.
- Janssen Vaccines and Prevention, Leiden, The Netherlands.
The peptides were constructed and purified by high-performance liquid chromatography (> 90% pure; CPC Scientific).
Zonari, A., Brace, L.E., Alencar-Silva, T., Porto, W.F., Foyt, D., Guiang, M., Cruz, E.A.O., Franco, O.L., Oliveira, C.R., Boroni, M. and Carvalho, J.L. Toxicology Reports 9 (2022): 1632-1638.
Peptide 14 (ETAKHWLKGI) (Sup. Fig. 1) was purchased from CPC Scientific Inc. (USA), which synthesized the peptide by solid phase (Fmoc) on a Rink amide resin, with > 95% purity, in the form of acetate salt.
CPC Scientific Inc., a leading global peptide CRDMO (Contract Research, Development, and Manufacturing Organization) has invested in a new peptide API (Active Pharmaceutical Ingredient) manufacturing site, bringing many new jobs to Rocklin, California. The 41,000 sq ft facility located at 3880 Atherton Rd, Rocklin, CA 95765 will be utilized to manufacture clinical to commercial grade peptide products for increased manufacturing capacity and will diversify CPC Scientific’s supply chain.
CPC Scientific is entering an exciting period of growth and innovation for peptide and oligonucleotide therapeutic development and manufacturing, and we will continue to provide therapeutic APIs to pharmaceutical and biotech companies around the world. We are very pleased to partner with the City of Rocklin, California to bring manufacturing and Life-Science jobs to local American workers,” said Shawn Lee, PhD, CEO.