Chapter 2
Production of Multiprotein Complexes Using
the Baculovirus Expression System: Homology-Based
and Restriction-Free Cloning Strategies for Construct
Design
Paola Rossolillo, Olga Kolesnikova, Karim Essabri,
Gala Ramon Zamorano, and Arnaud Poterszman
Abstract
Most cellular processes are mediated by multi-subunit protein complexes which have attracted major
interest in both academia and industry. Recombinant production of such entities in quantity and quality
sufficient for functional and structural investigations may be extremely challenging and necessitate specific
technologies. The baculovirus expression vector system is widely used for the production of eukaryotic
multiprotein complexes, and a variety of strategies are available to assemble transfer vectors for the
generation of recombinant baculoviruses. Here we detail applications of homology-based cloning techniques for one-step construction of dual promoter baculovirus transfer plasmids and of restriction-free
(RF) cloning for the modification of existing constructs.
Key words Baculovirus insect cell expression, Multiprotein complex, Homology-based cloning,
Sequence and ligation–independent cloning (SLIC), restriction-free (RF) cloning
1 Introduction
Most processes in living systems are mediated by multi-subunit
protein complexes which have attracted major interest in both
academia and industry. Recombinant production of such entities
in quantity and quality sufficient for functional and structural
investigations requires specific technologies. In favorable cases,
components of a complex can be expressed separately and assembled in vitro. This approach is unfortunately not always applicable
as isolated components of a complex are often poorly soluble or
misfolded in the absence of interacting partners. Complex samples,
including self-assembling multi-protein complexes, proteins requiring specific interacting partners, or post-translational modifications
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
17
Production of Multiprotein Complexes Using
the Baculovirus Expression System: Homology-Based
and Restriction-Free Cloning Strategies for Construct
Design
Paola Rossolillo, Olga Kolesnikova, Karim Essabri,
Gala Ramon Zamorano, and Arnaud Poterszman
Abstract
Most cellular processes are mediated by multi-subunit protein complexes which have attracted major
interest in both academia and industry. Recombinant production of such entities in quantity and quality
sufficient for functional and structural investigations may be extremely challenging and necessitate specific
technologies. The baculovirus expression vector system is widely used for the production of eukaryotic
multiprotein complexes, and a variety of strategies are available to assemble transfer vectors for the
generation of recombinant baculoviruses. Here we detail applications of homology-based cloning techniques for one-step construction of dual promoter baculovirus transfer plasmids and of restriction-free
(RF) cloning for the modification of existing constructs.
Key words Baculovirus insect cell expression, Multiprotein complex, Homology-based cloning,
Sequence and ligation–independent cloning (SLIC), restriction-free (RF) cloning
1 Introduction
Most processes in living systems are mediated by multi-subunit
protein complexes which have attracted major interest in both
academia and industry. Recombinant production of such entities
in quantity and quality sufficient for functional and structural
investigations requires specific technologies. In favorable cases,
components of a complex can be expressed separately and assembled in vitro. This approach is unfortunately not always applicable
as isolated components of a complex are often poorly soluble or
misfolded in the absence of interacting partners. Complex samples,
including self-assembling multi-protein complexes, proteins requiring specific interacting partners, or post-translational modifications
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
17