PERMutation Using Transposase Engineering (PERMUTE): A Simple Approach for Constructing Circularly Permuted Protein Libraries

  • Alicia M. Jones
  • Joshua T. Atkinson
  • Jonathan J. SilbergEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1498)


Rearrangements that alter the order of a protein’s sequence are used in the lab to study protein folding, improve activity, and build molecular switches. One of the simplest ways to rearrange a protein sequence is through random circular permutation, where native protein termini are linked together and new termini are created elsewhere through random backbone fission. Transposase mutagenesis has emerged as a simple way to generate libraries encoding different circularly permuted variants of proteins. With this approach, a synthetic transposon (called a permuteposon) is randomly inserted throughout a circularized gene to generate vectors that express different permuted variants of a protein. In this chapter, we outline the protocol for constructing combinatorial libraries of circularly permuted proteins using transposase mutagenesis, and we describe the different permuteposons that have been developed to facilitate library construction.

Key words

Circular permutation Library Protein engineering Transposase Transposon 



This work was supported by the National Science Foundation (1150138). AMJ and JTA were supported by the National Science Foundation Graduate Research Fellowship Program (NSF GFRP) under grant number (R3E821).


  1. 1.
    Ostermeier M (2009) Designing switchable enzymes. Curr Opin Struct Biol 19:442–448CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Yu Y, Lutz S (2011) Circular permutation: a different way to engineer enzyme structure and function. Trends Biotechnol 29:18–25CrossRefPubMedGoogle Scholar
  3. 3.
    Liberles DA, Teichmann SA, Bahar I, Bastolla U, Bloom J, Bornberg-Bauer E, Colwell LJ, de Koning APJ, Dokholyan NV, Echave J et al (2012) The interface of protein structure, protein biophysics, and molecular evolution. Protein Sci 21:769–785CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zhao H, Arnold FH (1997) Combinatorial protein design: strategies for screening protein libraries. Curr Opin Struct Biol 7:480–485CrossRefPubMedGoogle Scholar
  5. 5.
    Graf R, Schachman HK (1996) Random circular permutation of genes and expressed polypeptide chains: application of the method to the catalytic chains of aspartate transcarbamoylase. Proc Natl Acad Sci U S A 93:11591–11596CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hennecke J, Sebbel P, Glockshuber R (1999) Random circular permutation of DsbA reveals segments that are essential for protein folding and stability. J Mol Biol 286:1197–1215CrossRefPubMedGoogle Scholar
  7. 7.
    Guntas G, Ostermeier M (2004) Creation of an allosteric enzyme by domain insertion. J Mol Biol 336:263–273CrossRefPubMedGoogle Scholar
  8. 8.
    Hida K, Won SY, Di Pasquale G, Hanes J, Chiorini JA, Ostermeier M (2010) Sites in the AAV5 capsid tolerant to deletions and tandem duplications. Arch Biochem Biophys 496:1–8CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Judd J, Wei F, Nguyen PQ, Tartaglia LJ, Agbandje-McKenna M, Silberg JJ, Suh J (2012) Random insertion of mCherry into VP3 domain of adeno-associated virus yields fluorescent capsids with no loss of infectivity. Mol Ther Nucleic Acids 1:e54CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Haapa S, Taira S, Heikkinen E, Savilahti H (1999) An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications. Nucleic Acids Res 27:2777–2784CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Jones DD (2005) Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 beta-lactamase to an amino acid deletion. Nucleic Acids Res 33:e80CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Segall-Shapiro TH, Nguyen PQ, Dos Santos ED, Subedi S, Judd J, Suh J, Silberg JJ (2011) Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation. J Mol Biol 406:135–148CrossRefPubMedGoogle Scholar
  13. 13.
    Segall-Shapiro TH, Meyer AJ, Ellington AD, Sontag ED, Voigt CA (2014) A ‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase. Mol Syst Biol 10:742CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pandey N, Nobles CL, Zechiedrich L, Maresso AW, Silberg JJ (2014) Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions. ACS Synth Biol. 4:615–624. doi: 10.1021/sb5002938
  15. 15.
    Poussu E, Vihinen M, Paulin L, Savilahti H (2004) Probing the alpha-complementing domain of E. coli beta-galactosidase with use of an insertional pentapeptide mutagenesis strategy based on Mu in vitro DNA transposition. Proteins 54:681–692CrossRefPubMedGoogle Scholar
  16. 16.
    Edwards WR, Busse K, Allemann RK, Jone DD (2008) Linking the functions of unrelated proteins using a novel directed evolution domain insertion method. Nucleic Acids Res 36:e78CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Hoeller BM, Reiter B, Abad S, Graze I, Glieder A (2008) Random tag insertions by Transposon Integration mediated Mutagenesis (TIM). J Microbiol Methods 75:251–257CrossRefPubMedGoogle Scholar
  18. 18.
    Poussu E, Jäntti J, Savilahti H (2005) A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach. Nucleic Acids Res 33:e104CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Mehta MM, Liu S, Silberg JJ (2012) A transposase strategy for creating libraries of circularly permuted proteins. Nucleic Acids Res 40:e71CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Pandey N, Kuypers BE, Nassif B, Thomas EE, Alnahhas RN, Segatori L, Silberg JJ (2016) Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation. Biochem 55:3763–3773Google Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Alicia M. Jones
    • 1
  • Joshua T. Atkinson
    • 2
  • Jonathan J. Silberg
    • 1
    Email author
  1. 1.Biosciences DepartmentRice UniversityHoustonUSA
  2. 2.Systems, Synthetic, and Physical Biology Graduate ProgramRice UniversityHoustonUSA

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