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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Alberts B (1998) The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92(3):291–294
Luckow VA, Lee SC, Barry GF, Olins PO (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67(8):4566–4579
Bieniossek C, Imasaki T, Takagi Y, Berger I (2012) MultiBac: expanding the research toolbox for multiprotein complexes. Trends Biochem Sci 37(2):49–57. https://doi.org/10.1016/j.tibs.2011.10.005
Berger I, Fitzgerald DJ, Richmond TJ (2004) Baculovirus expression system for heterologous multiprotein complexes. Nat Biotechnol 22(12):1583–1587. https://doi.org/10.1038/nbt1036
Fitzgerald DJ, Berger P, Schaffitzel C, Yamada K, Richmond TJ, Berger I (2006) Protein complex expression by using multigene baculoviral vectors. Nat Methods 3(12):1021–1032. https://doi.org/10.1038/nmeth983
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(2):198–208. https://doi.org/10.1016/j.jsb.2011.03.007
Zhang Z, Yang J, Barford D (2016) Recombinant expression and reconstitution of multiprotein complexes by the USER cloning method in the insect cell-baculovirus expression system. Methods 95:13–25. https://doi.org/10.1016/j.ymeth.2015.10.003
Fitzgerald DJ, Schaffitzel C, Berger P, Wellinger R, Bieniossek C, Richmond TJ, Berger I (2007) Multiprotein expression strategy for structural biology of eukaryotic complexes. Structure 15(3):275–279. https://doi.org/10.1016/j.str.2007.01.016
Weissmann F, Petzold G, VanderLinden R, Huis In ‘t Veld PJ, Brown NG, Lampert F, Westermann S, Stark H, Schulman BA, Peters JM (2016) biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes. Proc Natl Acad Sci U S A 113(19):E2564–E2569. https://doi.org/10.1073/pnas.1604935113
Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA III, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. https://doi.org/10.1038/nmeth.1318
Qiao R, Weissmann F, Yamaguchi M, Brown NG, VanderLinden R, Imre R, Jarvis MA, Brunner MR, Davidson IF, Litos G, Haselbach D, Mechtler K, Stark H, Schulman BA, Peters JM (2016) Mechanism of APC/CCDC20 activation by mitotic phosphorylation. Proc Natl Acad Sci U S A 113(19):E2570–E2578. https://doi.org/10.1073/pnas.1604929113
Yamaguchi M, VanderLinden R, Weissmann F, Qiao R, Dube P, Brown NG, Haselbach D, Zhang W, Sidhu SS, Peters JM, Stark H, Schulman BA (2016) Cryo-EM of mitotic checkpoint complex-bound APC/C reveals reciprocal and conformational regulation of ubiquitin ligation. Mol Cell 63(4):593–607. https://doi.org/10.1016/j.molcel.2016.07.003
Brown NG, VanderLinden R, Watson ER, Weissmann F, Ordureau A, Wu KP, Zhang W, Yu S, Mercredi PY, Harrison JS, Davidson IF, Qiao R, Lu Y, Dube P, Brunner MR, Grace CRR, Miller DJ, Haselbach D, Jarvis MA, Yamaguchi M, Yanishevski D, Petzold G, Sidhu SS, Kuhlman B, Kirschner MW, Harper JW, Peters JM, Stark H, Schulman BA (2016) Dual RING E3 architectures regulate multiubiquitination and ubiquitin chain elongation by APC/C. Cell 165(6):1440–1453. https://doi.org/10.1016/j.cell.2016.05.037
Trowitzsch S, Bieniossek C, Nie Y, Garzoni F, Berger I (2010) New baculovirus expression tools for recombinant protein complex production. J Struct Biol 172(1):45–54. https://doi.org/10.1016/j.jsb.2010.02.010
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Weissmann, F., Peters, JM. (2018). Expressing Multi-subunit Complexes Using biGBac. In: Marsh, J. (eds) Protein Complex Assembly. Methods in Molecular Biology, vol 1764. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7759-8_21
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7759-8_21
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7758-1
Online ISBN: 978-1-4939-7759-8
eBook Packages: Springer Protocols