biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes.
Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular "mix and match" approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Florian
F
Weissmann
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Georg
G
Petzold
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105; Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, TN 38105;
Ryan
R
VanderLinden
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Pim J
PJ
Huis In 't Veld
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
Nicholas G
NG
Brown
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Fabienne
F
Lampert
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Stefan
S
Westermann
Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; Department of 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universität, 37077 Göttingen, Germany;
Holger
H
Stark
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105; Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, TN 38105; Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Sciences Center, Memphis, TN 38163 Brenda.Schulman@stjude.org Jan-Michael.Peters@imp.ac.at.
Brenda A
BA
Schulman
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria; Brenda.Schulman@stjude.org Jan-Michael.Peters@imp.ac.at.
Jan-Michael
JM
Peters
D016367:Baculoviridae / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D005810:Multigene Family / Q000235:genetics*; D046912:Multiprotein Complexes / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D046912:Multiprotein Complexes; D021122:Protein Subunits; D011994:Recombinant Proteins; D014764:Viral Proteins
Gibson assembly; baculovirus-insect cell expression; protein complexes