Trypsiligase-Catalyzed Peptide and Protein Ligation

  • Sandra Liebscher
  • Frank BordusaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2012)


Site-specific incorporation of nonproteinogenic functionalities into protein targets is an important tool in both basic and applied research and represents a major challenge to protein chemists. Chemical labeling methods often target multiple positions within a protein and therefore suffer from a lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this chapter we describe the application of the highly specific trypsin variant trypsiligase for the site-specific modification of virtual any target protein. We present two general routes of modification resulting in either N- or C-terminal functionalized protein products. Reactions rapidly proceed under mild conditions and result in homogeneously modified proteins bearing the artificial functionality exclusively at the desired position. We detail protocols for the expression and purification of trypsiligase as well as the synthesis of peptide (ester) substrates. In addition, we provide instructions for the bioconjugation reactions and for the qualitative and quantitative analysis of reaction progress and efficiency.

Key words

Trypsin variant Peptide ligation Protein modification Substrate-assisted catalysis Substrate mimetic Transpeptidation Trypsiligase 


  1. 1.
    Rademann J (2004) Organic protein chemistry: drug discovery through the chemical modification of proteins. Angew Chem Int Ed 43(35):4554–4556. Scholar
  2. 2.
    Stephanopoulos N, Francis MB (2011) Choosing an effective protein bioconjugation strategy. Nat Chem Biol 7:876. Scholar
  3. 3.
    Schmidt M, Toplak A, Quaedflieg PJLM et al (2017) Enzyme-mediated ligation technologies for peptides and proteins. Curr Opin Chem Biol 38:1–7. Scholar
  4. 4.
    Zhang Y, Park K-Y, Suazo KF et al (2018) Recent progress in enzymatic protein labelling techniques and their applications. Chem Soc Rev 47:9106. Scholar
  5. 5.
    Rush JS, Bertozzi CR (2008) New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo. J Am Chem Soc 130(37):12240–12241. Scholar
  6. 6.
    Radisky ES, Lee JM, Lu CJ et al (2006) Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates. Proc Natl Acad Sci U S A 103(18):6835–6840. Scholar
  7. 7.
    Hartley BS, Shotton DM, Paul DB (1971) 10 Pancreatic elastase. In: The enzymes, vol 3. Academic Press, London, pp 323–373. Scholar
  8. 8.
    Graf L, Craik CS, Patthy A et al (1987) Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin. Biochemistry 26(9):2616–2623. Scholar
  9. 9.
    Kurth T, Grahn S, Thormann M et al (1998) Engineering the S1′ subsite of trypsin: design of a protease which cleaves between dibasic residues. Biochemistry 37(33):11434–11440. Scholar
  10. 10.
    Willett WS, Brinen LS, Fletterick RJ et al (1996) Delocalizing trypsin specificity with metal activation. Biochemistry 35(19):5992–5998. Scholar
  11. 11.
    Liebscher S, Schoepfel M, Aumueller T et al (2014) N-terminal protein modification by substrate-activated reverse proteolysis. Angew Chem Int Ed 53(11):3024–3028. Scholar
  12. 12.
    Schumacher D, Helma J, Mann FA et al (2015) Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase. Angew Chem Int Ed 54(46):13787–13791. Scholar
  13. 13.
    Rashidian M, Dozier JK, Distefano MD (2013) Enzymatic labeling of proteins: techniques and approaches. Bioconjug Chem 24(8):1277–1294. Scholar
  14. 14.
    Altschul SF, Madden TL, Schaffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402. Scholar
  15. 15.
    Bordusa F (2002) Proteases in organic synthesis. Chem Rev 102(12):4817–4867. Scholar
  16. 16.
    Meyer C, Liebscher S, Bordusa F (2016) Selective coupling of click anchors to proteins via trypsiligase. Bioconjug Chem 27(1):47–53. Scholar
  17. 17.
    Liebscher S, Kornberger P, Fink G et al (2014) Derivatization of antibody Fab fragments: a designer enzyme for native protein modification. ChemBioChem 15(8):1096–1100. Scholar
  18. 18.
    Higgins DR, Cregg JM (1998) Introduction to Pichia pastoris. Methods Mol Biol 103:1–15. Scholar
  19. 19.
    Lal B, Gangopadhyay AK (1996) A practical synthesis of free and protected guanidino acids from amino acids. Tetrahedron Lett 37(14):2483–2486. Scholar
  20. 20.
    Sekizaki H, Itoh K, Toyota E et al (1996) Synthesis and triptic hydrolysis of p-guanidinophenyl esters derived from amino acids and peptides. Chem Pharm Bull(Tokyo) 44(8):1577–1579. Scholar
  21. 21.
    Wang H, Yang C, Wang L et al (2011) Self-assembled nanospheres as a novel delivery system for taxol: a molecular hydrogel with nanosphere morphology. Chem Commun 47(15):4439–4441. Scholar
  22. 22.
    Tokmina-Roszyk M, Tokmina-Roszyk D, Fields GB (2013) The synthesis and application of Fmoc-Lys(5-Fam) building blocks. Pept Sci 100(4):347–355. Scholar
  23. 23.
    Hoyle CE, Lowe AB, Bowman CN (2010) Thiol-click chemistry: a multifaceted toolbox for small molecule and polymer synthesis. Chem Soc Rev 39(4):1355–1387. Scholar
  24. 24.
    Schmidt TG, Skerra A (2007) The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc 2(6):1528–1535. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Biochemistry/Biotechnology, Charles-Tanford-Protein CenterMartin-Luther-University Halle-WittenbergHalleGermany

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