Expressing Multi-subunit Complexes Using biGBac

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1764)

Abstract

The reconstitution of recombinant protein complexes is facilitated by methods that allow coexpression of their subunits from a single vector. Here we describe a detailed step-by-step protocol for the biGBac cloning method which can be used to generate baculoviral transfer vectors coding for up to 25 subunits of a protein complex (Weissmann et al., Proc Natl Acad Sci U S A 113(19):E2564–E2569, 2016). biGBac is based on Gibson assembly reactions, optimized DNA linker sequences, and uses a hierarchical two-step assembly procedure. In the first assembly step, up to five expression cassettes are combined to generate a polygene cassette. In the second step, up to five polygene cassettes can then be combined to generate transfer vectors coding for up to 25 subunits.

Key words

Protein complex Baculovirus-insect cell expression BEVS Multigene expression Gibson assembly 

Notes

Acknowledgments

We would like to thank Georg Petzold and Brenda Schulman and her laboratory members for their invaluable contributions during development and validation of the biGBac technique. Research in the laboratory of J.-M.P. is supported by Boehringer Ingelheim, the Austrian Science Fund (SFB-F34 and Wittgenstein award Z196-B20), the Austrian Research Promotion Agency (headquarter grants FFG-834223 and FFG-852936, Laura Bassi Centre for Optimized Structural Studies grant FFG-840283), and the European Union (Seventh Framework Programme Grant 227764 MitoSys).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Research Institute of Molecular Pathology (IMP)Vienna Biocenter (VBC)ViennaAustria

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