Construction of Protein Switches by Domain Insertion and Directed Evolution

  • Lucas F. Ribeiro
  • Tiana D. Warren
  • Marc Ostermeier
Part of the Methods in Molecular Biology book series (MIMB, volume 1596)


A protein switch is a protein that changes between inactive (“off”) and active (“on”) states in response to a biomolecule or physical signal. These switches can be created by fusing two domains in such a way that the activity of the output domain is regulated by the input domain’s recognition of an input signal (such as the binding of a molecule, recognition of light). Here, we describe several methods for randomly fusing two domains to create domain insertion libraries from which protein switches can be identified by selections and/or screens.

Key words

Protein switch Domain insertion Circular permutation Directed evolution 


  1. 1.
    Wright CM, Wright RC, Eshleman JR, Ostermeier M (2011) A protein therapeutic modality founded on molecular regulation. Proc Natl Acad Sci U S A 108(39):16206–16211CrossRefGoogle Scholar
  2. 2.
    Alicea I, Marvin JS, Miklos AE, Ellington AD, Looger LL, Schreiter ER (2011) Structure of the Escherichia coli phosphonate binding protein PhnD and rationally optimized phosphonate biosensors. J Mol Biol 414(3):356–369CrossRefGoogle Scholar
  3. 3.
    Deuschle K, Fehr M, Hilpert M, Lager I, Lalonde S, Looger LL, Okumoto S, Persson J, Schmidt A, Frommer WB (2005) Genetically encoded sensors for metabolites. Cytometry A 64(1):3–9CrossRefGoogle Scholar
  4. 4.
    Deuschle K, Okumoto S, Fehr M, Looger LL, Kozhukh L, Frommer WB (2005) Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering. Protein Sci 14(9):2304–2314CrossRefGoogle Scholar
  5. 5.
    Ribeiro LF, Nicholes N, Tullman J, Ribeiro LFC, Fuzo CA, Vieira DS, Furtado GP, Ostermeier M, Ward RJ (2015) Insertion of a xylanase in xylose binding protein results in a xylose-stimulated xylanase. Biotechnol Biofuels 8:118Google Scholar
  6. 6.
    Dagliyan O, Shirvanyants D, Karginov AV, Ding F, Fee L, Chandrasekaran SN, Freisinger CM, Smolen GA, Huttenlocher A, Hahn KM, Dokholyan NV (2013) Rational design of a ligand-controlled protein conformational switch. Proc Natl Acad Sci U S A 110(17):6800–6804CrossRefGoogle Scholar
  7. 7.
    Guntas G, Mansell TJ, Kim JR, Ostermeier M (2005) Directed evolution of protein switches and their application to the creation of ligand-binding proteins. Proc Natl Acad Sci U S A 102(32):11224–11229CrossRefGoogle Scholar
  8. 8.
    Tullman J, Guntas G, Dumont M, Ostermeier M (2011) Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA. Biotechnol Bioeng 108(11):2535–2543Google Scholar
  9. 9.
    Tullman J, Nicholes N, Dumont MR, Ribeiro LF, Ostermeier M (2015) Enzymatic protein switches built from paralogous input domains. Biotechnol Bioeng 9999:1–7Google Scholar
  10. 10.
    Tullman J, Guntas G, Dumont M, Ostermeier M (2011) Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA. Biotechnol Bioeng 108(11):2535–2543CrossRefGoogle Scholar
  11. 11.
    Guntas G, Mitchell SF, Ostermeier M (2004) A molecular switch created by in vitro recombination of nonhomologous genes. Chem Biol 11(11):1483–1487CrossRefGoogle Scholar
  12. 12.
    Guntas G, Ostermeier M (2004) Creation of an allosteric enzyme by domain insertion. J Mol Biol 336(1):263–273CrossRefGoogle Scholar
  13. 13.
    Ostermeier M, Guntas G (2003) Engineering a protein molecular switch by combinatorial domain insertion. Abstr Pap Am Chem S 225:U243–U243Google Scholar
  14. 14.
    Guntas G, Kanwar M, Ostermeier M (2012) Circular permutation in the omega-loop of TEM-1 beta-lactamase results in improved activity and altered substrate specificity. PLoS One 7(4):e35998CrossRefGoogle Scholar
  15. 15.
    Nicholes N, Date A, Beaujean P, Hauk P, Kanwar M, Ostermeier M (2016) Modular protein switches derived from antibody mimetic proteins. Protein Eng Des Sel 29(2):77–85CrossRefGoogle Scholar
  16. 16.
    Choi JH, Laurent AH, Hilser VJ, Ostermeier M (2015) Design of protein switches based on an ensemble model of allostery. Nat Commun 6: 6968Google Scholar
  17. 17.
    Choi JH, Zayats M, Searson PC, Ostermeier M (2016) Electrochemical activation of engineered protein switches. Biotechnol Bioeng 113(2):453–456CrossRefGoogle Scholar
  18. 18.
    Heins RA, Choi JH, Sohka T, Ostermeier M (2011) In vitro recombination of non-homologous genes can result in gene fusions that confer a switching phenotype to cells. PLoS One 6(11):e27302CrossRefGoogle Scholar
  19. 19.
    Yu Y, Lutz S (2011) Circular permutation: a different way to engineer enzyme structure and function. Trends Biotechnol 29(1):18–25CrossRefGoogle Scholar
  20. 20.
    Bosley AD, Ostermeier M (2005) Mathematical expressions useful in the construction, description and evaluation of protein libraries. Biomol Eng 22(1–3):57–61CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Lucas F. Ribeiro
    • 1
  • Tiana D. Warren
    • 1
  • Marc Ostermeier
    • 1
  1. 1.Department of Chemical and Biomolecular EngineeringJohns Hopkins UniversityBaltimoreUSA

Personalised recommendations