Application of In-Fusion™ Cloning for the Parallel Construction of E. coli Expression Vectors

  • Louise E. Bird
  • Heather Rada
  • John Flanagan
  • Jonathan M. Diprose
  • Robert J. C. Gilbert
  • Raymond J. Owens
Part of the Methods in Molecular Biology book series (MIMB, volume 1116)


In-Fusion™ cloning is a flexible DNA ligase-independent cloning technology that has wide-ranging uses in molecular biology. In this chapter we describe the protocols used in the OPPF-UK to design and construct expression vectors using In-Fusion™. Our method for small scale expression screening in Escherichia coli of constructs generated by In-Fusion™ is also outlined.

Key words

In-Fusion™ enzyme PCR cloning Single-stranded annealing Fusion tags Co-expression Periplasmic secretion Escherichia coli 



The Oxford Protein Production Facility-UK is supported by the UK Medical Research Council and the Biotechnology and Biology Research Council (MRC Grant MR/K018779/1). We thank Jo Nettleship for helpful discussions on E. coli secretion vectors.


  1. 1.
    Berrow NS, Bussow K, Coutard B et al (2006) Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta Crystallogr D Biol Crystallogr 62: 1218–1226PubMedCrossRefGoogle Scholar
  2. 2.
    Savitsky P, Bray J, Cooper CD et al (2010) High-throughput production of human proteins for crystallization: the SGC experience. J Struct Biol 172:3–13PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Graslund S, Nordlund P, Weigelt J et al (2008) Protein production and purification. Nat Methods 5:135–146PubMedCrossRefGoogle Scholar
  4. 4.
    Irwin CR, Farmer A, Willer DO et al (2012) In-fusion(R) cloning with vaccinia virus DNA polymerase. Methods Mol Biol 890:23–35PubMedCrossRefGoogle Scholar
  5. 5.
    Hamilton MD, Nuara AA, Gammon DB et al (2007) Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Res 35:143–151PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Aslandis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074CrossRefGoogle Scholar
  7. 7.
    Haun RS, Serventi IM, Moss J (1992) Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors. Biotechniques 13:515–518PubMedGoogle Scholar
  8. 8.
    Li MZ, Elledge SJ (2012) SLIC: a method for sequence- and ligation-independent cloning. Methods Mol Biol 852:51–59PubMedCrossRefGoogle Scholar
  9. 9.
    Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4: 251–256PubMedCrossRefGoogle Scholar
  10. 10.
    Jeong JY, Yim HS, Ryu JY et al (2012) One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl Environ Microbiol 78:5440–5443PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Gibson DG, Young L, Chuang RY et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6: 343–345PubMedCrossRefGoogle Scholar
  12. 12.
    Gibson DG, Smith HO, Hutchison CA III et al (2010) Chemical synthesis of the mouse mitochondrial genome. Nat Methods 7:901–903PubMedCrossRefGoogle Scholar
  13. 13.
    Berrow NS, Alderton D, Sainsbury S et al (2007) A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res 35:e45PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Berrow NS, Alderton D, Owens RJ (2009) The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning. Methods Mol Biol 498:75–90PubMedCrossRefGoogle Scholar
  15. 15.
    Bird LE (2011) High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli. Methods 55:29–37PubMedCrossRefGoogle Scholar
  16. 16.
    Chen JH, Jung JW, Wang Y et al (2010) Immunoproteomics profiling of blood stage Plasmodium vivax infection by high-throughput screening assays. J Proteome Res 9:6479–6489PubMedCrossRefGoogle Scholar
  17. 17.
    Howland SW, Poh CM, Renia L (2011) Directional, seamless, and restriction enzyme-free construction of random-primed complementary DNA libraries using phosphorothioate-modified primers. Anal Biochem 416:141–143PubMedCrossRefGoogle Scholar
  18. 18.
    Nettleship JE, Ren J, Rahman N et al (2008) A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells. Protein Expr Purif 62:83–89PubMedCrossRefGoogle Scholar
  19. 19.
    Au K, Berrow NS, Blagova E et al (2006) Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis. Acta Crystallogr D Biol Crystallogr 62:1267–1275PubMedCrossRefGoogle Scholar
  20. 20.
    Marsischky G, LaBaer J (2004) Many paths to many clones: a comparative look at high-throughput cloning methods. Genome Res 14:2020–2028PubMedCrossRefGoogle Scholar
  21. 21.
    Zhu B, Cai G, Hall EO et al (2007) In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43:354–359PubMedCrossRefGoogle Scholar
  22. 22.
    Zhou B, Donnelly ME, Scholes DT et al (2009) Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and Swine origin human influenza a viruses. J Virol 83:10309–10313PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Sleight SC, Bartley BA, Lieviant JA et al (2010) Designing and engineering evolutionary robust genetic circuits. J Biol Eng 4:12PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Sleight SC, Bartley BA, Lieviant JA et al (2010) In-Fusion BioBrick assembly and re-engineering. Nucleic Acids Res 38:2624–2636PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Alzari PM, Berglund H, Berrow NS et al (2006) Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. Acta Crystallogr D Biol Crystallogr 62:1103–1113PubMedCrossRefGoogle Scholar
  26. 26.
    Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17:353–358PubMedCrossRefGoogle Scholar
  27. 27.
    Marblestone JG, Edavettal SC, Lim Y et al (2006) Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. Protein Sci 15:182–189PubMedCrossRefGoogle Scholar
  28. 28.
    Ohana RF, Encell LP, Zhao K et al (2009) HaloTag7: a genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification. Protein Expr Purif 68:110–120PubMedCrossRefGoogle Scholar
  29. 29.
    Chudakov DM, Matz MV, Lukyanov S et al (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90:1103–1163PubMedCrossRefGoogle Scholar
  30. 30.
    Li C, Schwabe JW, Banayo E et al (1997) Coexpression of nuclear receptor partners increases their solubility and biological activities. Proc Natl Acad Sci U S A 94:2278–2283PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Alexandrov A, Vignali M, LaCount DJ et al (2004) A facile method for high-throughput co-expression of protein pairs. Mol Cell Proteomics 3:934–938PubMedCrossRefGoogle Scholar
  32. 32.
    Scheich C, Kummel D, Soumailakakis D et al (2007) Vectors for co-expression of an unrestricted number of proteins. Nucleic Acids Res 35:e43PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Kholod N, Mustelin T (2001) Novel vectors for co-expression of two proteins in E. coli. Biotechniques 31(322–323):326–328Google Scholar
  34. 34.
    Yang W, Zhang L, Lu Z et al (2001) A new method for protein coexpression in Escherichia coli using two incompatible plasmids. Protein Expr Purif 22:472–478PubMedCrossRefGoogle Scholar
  35. 35.
    Hinnebusch AG (2006) eIF3: a versatile scaffold for translation initiation complexes. Trends Biochem Sci 31:553–562PubMedCrossRefGoogle Scholar
  36. 36.
    Busso D, Peleg Y, Heidebrecht T et al (2011) Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study. J Struct Biol 175:159–170PubMedCrossRefGoogle Scholar
  37. 37.
    Economou A, Christie PJ, Fernandez RC et al (2006) Secretion by numbers: protein traffic in prokaryotes. Mol Microbiol 62:308–319PubMedCrossRefGoogle Scholar
  38. 38.
    Desvaux M, Hebraud M, Talon R et al (2009) Secretion and subcellular localizations of bacterial proteins: a semantic awareness issue. Trends Microbiol 17:139–145PubMedCrossRefGoogle Scholar
  39. 39.
    Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64:625–635PubMedCrossRefGoogle Scholar
  40. 40.
    Mergulhao FJ, Summers DK, Monteiro GA (2005) Recombinant protein secretion in Escherichia coli. Biotechnol Adv 23:177–202PubMedCrossRefGoogle Scholar
  41. 41.
    Binet R, Letoffe S, Ghigo JM et al (1997) Protein secretion by Gram-negative bacterial ABC exporters—a review. Gene 192:7–11PubMedCrossRefGoogle Scholar
  42. 42.
    Steiner D, Forrer P, Stumpp MT et al (2006) Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nat Biotechnol 24:823–831PubMedCrossRefGoogle Scholar
  43. 43.
    Benoit RM, Wilhelm RN, Scherer-Becker D et al (2006) An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids. Protein Expr Purif 45:66–71PubMedCrossRefGoogle Scholar
  44. 44.
    Ruther U (1980) Construction and properties of a new cloning vehicle, allowing direct screening for recombinant plasmids. Mol Gen Genet 178:475–477PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Louise E. Bird
    • 1
    • 2
  • Heather Rada
    • 1
    • 2
  • John Flanagan
    • 2
  • Jonathan M. Diprose
    • 1
    • 2
  • Robert J. C. Gilbert
    • 2
  • Raymond J. Owens
    • 1
    • 2
  1. 1.Oxford Protein Production Facility-UKOxfordshireUK
  2. 2.Division of Structural Biology, Henry Wellcome Building for Genomic MedicineUniversity of OxfordOxfordUK

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