Site-Specific Modification of Proteins via Trypsiligase

  • Sandra Liebscher
  • Frank BordusaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2033)


Site-specific incorporation of artificial functionalities into protein targets is an important tool in both basic and applied research and can be a major challenge to protein chemists. Chemical labeling methods often targeting multiple positions within a protein and therefore suffer from lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this contribution we describe the application of the highly specific trypsin variant named 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. Both reaction regimes proceed under mild and bioorthogonal conditions in a short period of time which 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 construction of peptide or acyl donor ester probes used as substrates for the biocatalyst. In addition, we provide instructions how to perform the ultimate bioconjugation reactions and finally render assistance for the qualitative and quantitative analysis of the reaction course and outcome.

Key words

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


  1. 1.
    Hartley BS, Shotton DM, Paul DB (1971) 10 pancreatic elastase. Enzymes 3:323–373. Scholar
  2. 2.
    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
  3. 3.
    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
  4. 4.
    Willett WS, Brinen LS, Fletterick RJ et al (1996) Delocalizing trypsin specificity with metal activation. Biochemistry 35(19):5992–5998. Scholar
  5. 5.
    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
  6. 6.
    Bordusa F (2002) Proteases in organic synthesis. Chem Rev 102(12):4817–4868. Scholar
  7. 7.
    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
  8. 8.
    Meyer C, Liebscher S, Bordusa F (2016) Selective coupling of click anchors to proteins via trypsiligase. Bioconjug Chem 27(1):47–53. Scholar
  9. 9.
    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 44(8):1577–1579. Scholar
  10. 10.
    Coin I, Beyermann M, Bienert M (2007) Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat Protoc 2:3247. Scholar
  11. 11.
    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
  12. 12.
    Higgins DR, Cregg JM (1998) Introduction to Pichia pastoris. Methods Mol Biol 103:1–15. Scholar
  13. 13.
    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
  14. 14.
    Bouvet J-P (1994) Immunoglobulin Fab fragment-binding proteins. Int J Immunopharmacol 16(5):419–424. Scholar
  15. 15.
    Skerra A (1994) Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151(1):131–135. Scholar
  16. 16.
    Skerra A (1993) Bacterial expression of immunoglobulin fragments. Curr Opin Immunol 5(2):256–262. Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Charles-Tanford-Protein Center, Institute of Biochemistry/BiotechnologyMartin-Luther-University Halle-WittenbergHalleGermany

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