Skip to main content
SpringerLink
Log in

Removable Backbone Modification (RBM) Strategy for the Chemical Synthesis of Hydrophobic Peptides/Proteins

  • Protocol
  • First Online:
Chemical Protein Synthesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2530))

  • 893 Accesses

Abstract

Chemical synthesis can provide hydrophobic proteins with natural or man-made modifications (e.g. S-palmitoylation, site-specific isotope labeling and mirror-image proteins) that are difficult to obtain through the recombinant expression technology. The difficulty of chemical synthesis of hydrophobic proteins stems from the hydrophobic nature. Removable backbone modificaiton (RBM) strategy has been developed for solubilizing the hydrophobic peptides/proteins. Here we take the chemical synthesis of a S-palmitoylated peptide as an example to describe the detailed procedure of RBM strategy. Three critical steps of this protocol are: (1) installation of Lys6-tagged RBM groups into the peptides by Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis, (2) chemical ligation of the peptides, and (3) removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to give the target peptide.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Huang Y-C, Fang G-M, Liu L (2016) Chemical synthesis of proteins using hydrazide intermediates. Nat Sci Rev 3(1):107–116. https://doi.org/10.1093/nsr/nwv072

    Article  CAS  Google Scholar 

  2. Kulkarni SS, Sayers J, Premdjee B, Payne RJ (2018) Rapid and efficient protein synthesis through expansion of the native chemical ligation concept. Nat Rev Chem 2:0122. https://doi.org/10.1038/s41570-018-0122

    Article  CAS  Google Scholar 

  3. Tan Y, Wu H, Wei T, Li X (2020) Chemical protein synthesis: advances, challenges, and outlooks. J Am Chem Soc 42(48):20288–20298. https://doi.org/10.1021/jacs.0c09664

    Article  CAS  Google Scholar 

  4. Hartrampf N, Saebi A, Poskus M, Gates ZP, Callahan AJ, Cowfer AE, Hanna S, Antilla S, Schissel CK, Quartararo AJ, Ye X, Mijalis AJ, Simon MD, Loas A, Liu S, Jessen C, Nielsen TE, Pentelute BL (2020) Synthesis of proteins by automated flow chemistry. Science 368(6494):980–987. https://doi.org/10.1126/science.abb2491

    Article  CAS  PubMed  Google Scholar 

  5. Dawson PE, Muir TW, Clark-Lewis I, Kent SB (1994) Synthesis of proteins by native chemical ligation. Science 266(5186):776–779

    Article  CAS  Google Scholar 

  6. Bode JW, Fox RM, Baucom KD (2006) Chemoselective amide ligations by decarboxylative condensations of N-alkylhydroxylamines and alpha-ketoacids. Angew Chem Int Ed 45(8):1248–1252. https://doi.org/10.1002/anie.200503991

    Article  CAS  Google Scholar 

  7. Li X, Lam HY, Zhang Y, Chan CK (2010) Salicylaldehyde ester-induced chemoselective peptide ligations: enabling generation of natural peptidic linkages at the serine/threonine sites. Org Lett 12(8):1724–1727. https://doi.org/10.1021/ol1003109

    Article  CAS  PubMed  Google Scholar 

  8. Fang GM, Li YM, Shen F, Huang YC, Li JB, Lin Y, Cui HK, Liu L (2011) Protein chemical synthesis by ligation of peptide hydrazides. Angew Chem Int Ed 50(33):7645–7649. https://doi.org/10.1002/anie.201100996

    Article  CAS  Google Scholar 

  9. Fang GM, Wang JX, Liu L (2012) Convergent chemical synthesis of proteins by ligation of peptide hydrazides. Angew Chem Int Ed 51(41):10347–10350. https://doi.org/10.1002/anie.201203843

    Article  CAS  Google Scholar 

  10. Tang S, Si YY, Wang ZP, Mei KR, Chen X, Cheng JY, Zheng JS, Liu L (2015) An efficient one-pot four-segment condensation method for protein chemical synthesis. Angew Chem Int Ed 54(19):5713–5717. https://doi.org/10.1002/anie.201500051

    Article  CAS  Google Scholar 

  11. Chisholm TS, Clayton D, Dowman LJ, Sayers J, Payne RJ (2018) Native chemical ligation-photodesulfurization in flow. J Am Chem Soc 140(29):9020–9024. https://doi.org/10.1021/jacs.8b03115

    Article  CAS  PubMed  Google Scholar 

  12. Zheng JS, Tang S, Huang YC, Liu L (2013) Development of new thioester equivalents for protein chemical synthesis. Acc Chem Res 46(11):2475–2484. https://doi.org/10.1021/ar400012w

    Article  CAS  PubMed  Google Scholar 

  13. Agouridas V, El Mahdi O, Diemer V, Cargoet M, Monbaliu JM, Melnyk O (2019) Native chemical ligation and extended methods: mechanisms, catalysis, scope, and limitations. Chem Rev 119(12):7328–7443. https://doi.org/10.1021/acs.chemrev.8b00712

    Article  CAS  PubMed  Google Scholar 

  14. Qi Y-K, Si Y-Y, Du S-S, Liang J, Wang K-W, Zheng J-S (2019) Recent advances in the chemical synthesis and semi-synthesis of poly-ubiquitin-based proteins and probes. Sci China Chem 62(3):299–312. https://doi.org/10.1007/s11426-018-9401-8

    Article  CAS  Google Scholar 

  15. Wang P, Dong S, Shieh JH, Peguero E, Hendrickson R, Moore MAS, Danishefsky SJ (2013) Erythropoietin derived by chemical synthesis. Science 342(6164):1357–1360. https://doi.org/10.1126/science.1245095

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dann GP, Liszczak GP, Bagert JD, Muller MM, Nguyen UTT, Wojcik F, Brown ZZ, Bos J, Panchenko T, Pihl R, Pollock SB, Diehl KL, Allis CD, Muir TW (2017) ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference. Nature 548(7669):607–611. https://doi.org/10.1038/nature23671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Qu Q, Gao S, Wu F, Zhang MG, Li Y, Zhang LH, Bierer D, Tian CL, Zheng JS, Liu L (2020) Synthesis of disulfide surrogate peptides incorporating large-span surrogate bridges through a native-chemical-ligation-assisted diaminodiacid strategy. Angew Chem Int Ed 59(15):6037–6045. https://doi.org/10.1002/anie.201915358

    Article  CAS  Google Scholar 

  18. Zhou X, Zuo C, Li W, Shi W, Zhou X, Wang H, Chen S, Du J, Chen G, Zhai W, Zhao W, Wu Y, Qi Y, Liu L, Gao Y (2020) A novel d-peptide identified by mirror-image phage display blocks TIGIT/PVR for cancer immunotherapy. Angew Chem Int Ed 59(35):15114–15118. https://doi.org/10.1002/anie.202002783

    Article  CAS  Google Scholar 

  19. Pan M, Gao S, Zheng Y, Tan X, Lan H, Tan X, Sun D, Lu L, Wang T, Zheng Q, Huang Y, Wang J, Liu L (2016) Quasi-racemic X-ray structures of K27-linked ubiquitin chains prepared by total chemical synthesis. J Am Chem Soc 138(23):7429–7435. https://doi.org/10.1021/jacs.6b04031

    Article  CAS  PubMed  Google Scholar 

  20. David Y, Muir TW (2017) Emerging chemistry strategies for engineering native chromatin. J Am Chem Soc 139(27):9090–9096. https://doi.org/10.1021/jacs.7b03430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Mulder MPC, Zhuang Z, Liu L, Kessler BM, Ovaa H (2020) Editorial: Probing the ubiquitin landscape. Front Chem 8:449. https://doi.org/10.3389/fchem.2020.00449

    Article  PubMed  PubMed Central  Google Scholar 

  22. Liang LJ, Chu GC, Qu Q, Zuo C, Mao J, Zheng Q, Chen J, Meng X, Jing Y, Deng H, Li YM, Liu L (2021) Chemical synthesis of activity-based E2-ubiquitin probes for the structural analysis of E3 ligase-catalyzed transthiolation. Angew Chem Int Ed 60(31):17171. https://doi.org/10.1002/anie.202105870

    Article  CAS  Google Scholar 

  23. Li Y, Cao X, Tian C, Zheng J-S (2020) Chemical protein synthesis-assisted high-throughput screening strategies for d-peptides in drug discovery. Chin Chem Lett 31(9):2365–2374. https://doi.org/10.1016/j.cclet.2020.04.015

    Article  CAS  Google Scholar 

  24. Wang Z, Xu W, Liu L, Zhu TF (2016) A synthetic molecular system capable of mirror-image genetic replication and transcription. Nat Chem 8(7):698–704. https://doi.org/10.1038/nchem.2517

    Article  CAS  PubMed  Google Scholar 

  25. Zheng J-S, Liang J, Shi W-W, Li Y, Hu H-G, Tian C-L, Liu L (2021) A mirror-image protein-based information barcoding and storage technology. Sci Bull 66(15):1542–1549. https://doi.org/10.1016/j.scib.2021.03.010

    Article  CAS  Google Scholar 

  26. Shen F, Tang S, Liu L (2011) Hexafluoro-2-propanol as a potent cosolvent for chemical ligation of membrane proteins. Sci China Chem 54(1):110–116. https://doi.org/10.1007/s11426-010-4188-4

    Article  CAS  Google Scholar 

  27. Lahiri S, Brehs M, Olschewski D, Becker CF (2011) Total chemical synthesis of an integral membrane enzyme: diacylglycerol kinase from Escherichia coli. Angew Chem Int Ed 50(17):3988–3992. https://doi.org/10.1002/anie.201006686

    Article  CAS  Google Scholar 

  28. Johnson EC, Kent SB (2007) Towards the total chemical synthesis of integral membrane proteins: a general method for the synthesis of hydrophobic peptide-thioester building blocks. Tetrahedron Lett 48(10):1795–1799. https://doi.org/10.1016/j.tetlet.2007.01.030

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jacobsen MT, Petersen ME, Ye X, Galibert M, Lorimer GH, Aucagne V, Kay MS (2016) A helping hand to overcome solubility challenges in chemical protein synthesis. J Am Chem Soc 138(36):11775–11782. https://doi.org/10.1021/jacs.6b05719

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Asahina Y, Kamitori S, Takao T, Nishi N, Hojo H (2013) Chemoenzymatic synthesis of the immunoglobulin domain of Tim-3 carrying a complex-type N-glycan by using a one-pot ligation. Angew Chem Int Ed 52(37):9733–9737. https://doi.org/10.1002/anie.201303073

    Article  CAS  Google Scholar 

  31. Zheng JS, Yu M, Qi YK, Tang S, Shen F, Wang ZP, Xiao L, Zhang L, Tian CL, Liu L (2014) Expedient total synthesis of small to medium-sized membrane proteins via Fmoc chemistry. J Am Chem Soc 136(9):3695–3704. https://doi.org/10.1021/ja500222u

    Article  CAS  PubMed  Google Scholar 

  32. Zheng JS, He Y, Zuo C, Cai XY, Tang S, Wang ZA, Zhang LH, Tian CL, Liu L (2016) Robust chemical synthesis of membrane proteins through a general method of removable backbone modification. J Am Chem Soc 138(10):3553–3561. https://doi.org/10.1021/jacs.6b00515

    Article  CAS  PubMed  Google Scholar 

  33. Huang DL, Montigny C, Zheng Y, Beswick V, Li Y, Cao XX, Barbot T, Jaxel C, Liang J, Xue M, Tian CL, Jamin N, Zheng JS (2020) Chemical synthesis of native S-palmitoylated membrane proteins through removable-backbone-modification-assisted Ser/Thr ligation. Angew Chem Int Ed 59(13):5178–5184. https://doi.org/10.1002/anie.201914836

    Article  CAS  Google Scholar 

  34. Huang DL, Li Y, Liang J, Yu L, Xue M, Cao XX, Xiao B, Tian CL, Liu L, Zheng JS (2020) The new salicylaldehyde S,S-propanedithioacetal ester enables N-to-C sequential native chemical ligation and Ser/Thr ligation for chemical protein synthesis. J Am Chem Soc 142(19):8790–8799. https://doi.org/10.1021/jacs.0c01561

    Article  CAS  PubMed  Google Scholar 

  35. Zuo C, Tang S, Zheng J-S (2015) Chemical synthesis and biophysical applications of membrane proteins. J Pept Sci 21(7):540–549. https://doi.org/10.1002/psc.2721

    Article  CAS  PubMed  Google Scholar 

  36. Li JB, Tang S, Zheng JS, Tian CL, Liu L (2017) Removable backbone modification method for the chemical synthesis of membrane proteins. Acc Chem Res 50(5):1143–1153. https://doi.org/10.1021/acs.accounts.7b00001

    Article  CAS  PubMed  Google Scholar 

  37. Zuo C, Tang S, Si Y-Y, Wang ZA, Tian C-L, Zheng J-S (2016) Efficient synthesis of longer A beta peptides via removable backbone modification. Org Biomol Chem 14(22):5012–5018. https://doi.org/10.1039/c6ob00712k

    Article  CAS  PubMed  Google Scholar 

  38. Guo QY, Zhang LH, Zuo C, Huang DL, Wang ZA, Zheng JS, Tian CL (2019) Channel activity of mirror-image M2 proton channel of influenza A virus is blocked by achiral or chiral inhibitors. Protein Cell 10(3):211–216. https://doi.org/10.1007/s13238-018-0536-5

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Key R&D Program of China (No. 2019YFA0706900), the National Natural Science Foundation of China (Nos. 22022703 and 22177108), and the Science and Technological Fund of Anhui Province for Outstanding Youth (1808085J04).

Author information

Authors and Affiliations

  1. The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui, China

    Dong-Liang Huang, Ying Li & Ji-Shen Zheng

Authors
  1. Dong-Liang Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ying Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ji-Shen Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ji-Shen Zheng .

Editor information

Editors and Affiliations

  1. Department of Chemistry, University of Hong Kong, Hong Kong, SAR, P. R, China

    Xuechen Li

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Huang, DL., Li, Y., Zheng, JS. (2022). Removable Backbone Modification (RBM) Strategy for the Chemical Synthesis of Hydrophobic Peptides/Proteins. In: Li, X. (eds) Chemical Protein Synthesis. Methods in Molecular Biology, vol 2530. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2489-0_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2489-0_16

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2488-3

  • Online ISBN: 978-1-0716-2489-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation