Tandem Recombineering by SLIC Cloning and Cre-LoxP Fusion to Generate Multigene Expression Constructs for Protein Complex Research

  • Matthias Haffke
  • Cristina Viola
  • Yan Nie
  • Imre Berger
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1073)

Abstract

A robust protocol to generate recombinant DNA containing multigene expression cassettes by using sequence and ligation independent cloning (SLIC) followed by multiplasmid Cre-LoxP recombination in tandem for multiprotein complex research is described. The protocol includes polymerase chain reaction (PCR) amplification of the desired genes, seamless insertion into the target vector via SLIC, and Cre-LoxP recombination of specific donor and acceptor plasmid molecules, optionally in a robotic setup. This procedure, called tandem recombineering, has been implemented for multiprotein expression in E. coli and mammalian cells, and also for insect cells using a recombinant baculovirus.

Key words

Sequence and ligation independent cloning Cre recombinase Cre-LoxP fusion Multigene delivery Multiprotein complexes MultiBac ACEMBL automation Robotics 

Notes

Acknowledgments

We thank all members of the Berger laboratory for helpful discussions. M.H. is recipient of a Kekulé fellowship of the Fonds der Chemischen Industrie (FCI, Germany). Y.N. is a fellow of the Marie-Curie training network Chromatin Plasticity and the Boehringer Ingelheim Foundation (BIF, Germany). I.B. acknowledges support from the Swiss National Science Foundation (SNSF), the Agence Nationale de la Recherche (ANR), the Centre National de la Recherche Scientifique (CNRS), the EMBL and the European Commission (EC) through the joint EIPOD program, and the European Commission (EC) projects SPINE2-Complexes and 3D-Repertoire (Framework Program 6 (FP6)), as well as INSTRUCT, PCUBE, BioSTRUCT-X, and ComplexINC (EC FP7).

Competing financial interest statement: The authors declare competing financial interests. I.B. is author on patents and patent applications related to the methods here described.

References

  1. 1.
    Ellis T, Adie T, Baldwin GS (2011) DNA assembly for synthetic biology: from parts to pathways and beyond. Integr Biol 3:109–118CrossRefGoogle Scholar
  2. 2.
    Scharf SJ, Horn GT, Erlich HA (1986) Direct cloning and sequencing analysis of enzymatically amplified genomic sequences. Science 233:1076–1078CrossRefGoogle Scholar
  3. 3.
    Costa GL, Grafsky A, Weiner MP (1994) Cloning and analysis of PCR-generated DNA fragments. PCR Methods Appl 3:338–345CrossRefGoogle Scholar
  4. 4.
    Esposito D, Garvey LA, Chakiath CS (2009) Gateway cloning for protein expression. Methods Mol Biol 498:31–54CrossRefGoogle Scholar
  5. 5.
    Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256CrossRefGoogle Scholar
  6. 6.
    Bieniossek C, Nie Y, Frey D, Olieric N, Schaffitzel C, Collinson I, Romier C, Berger P, Richmond TJ, Steinmetz MO, Berger I (2009) Automated unrestricted multigene recombineering for multiprotein complex production. Nat Methods 6:447–450CrossRefGoogle Scholar
  7. 7.
    Nie Y, Bieniossek C, Frey D, Olieric N, Schaffitzel C, Steinmetz MO, Berger I (2009) ACEMBLing multigene expression vectors by recombineering. Nat Protoc 4. doi: 10.1038/nprot.2009.104
  8. 8.
    Kriz A, Schmid K, Baumgartner N, Ziegler U, Berger I, Ballmer-Hofer K, Berger P (2010) A plasmid-based multigene expression system for mammalian cells. Nat Commun 1. doi: 10.1038/ncomms1120
  9. 9.
    Fitzgerald DJ, Berger P, Schaffitzel C, Yamada K, Richmond TJ, Berger I (2006) Protein complex expression by using multigene baculoviral vectors. Nat Methods 3:1021–1032CrossRefGoogle Scholar
  10. 10.
    Berger I, Fitzgerald DJ, Richmond TJ (2004) Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol 22:1583–1587CrossRefGoogle Scholar
  11. 11.
    Vijayachandran LS, Viola C, Garzoni F, Trowitzsch S, Bieniossek C, Chaillet M, Schaffitzel C, Busso D, Romier C, Poterszman A, Richmond TJ, Berger I (2011) Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. J Struct Biol 175:198–208CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Matthias Haffke
    • 1
    • 2
  • Cristina Viola
    • 1
    • 2
  • Yan Nie
    • 1
    • 2
  • Imre Berger
    • 1
    • 2
  1. 1.European Molecular Biology Laboratory (EMBL), BP 181Polygone ScientifiqueGrenobleFrance
  2. 2.Unit of Virus Host Cell Interactions (UVHCI)Polygone ScientifiqueGrenobleFrance

Personalised recommendations