frequently require the simultaneous expression of several proteins
foreign to the host cell.
With the baculovirus insect cell expression system, protein
targets can be expressed from multiple viruses carrying a single
foreign gene each, from a single baculovirus carrying multiple
foreign genes, or from a combination of the two approaches. For
routine production of complex systems, it is now widely accepted
that the use of multigene expression constructs is extremely efficient. Progress in our understanding of the baculovirus biology
together with the development of new tools for DNA manipulation
have allowed to streamline protocols for the generation of viruses,
expression screening, and large-scale productions (see refs. 1, 2 for
recent reviews). A major breakthrough was accomplished through
the development of the Multibac technology, which directs
co-expression of multiple genes from a single virus under the
control of multiple copies of the late p10 promoter and the very
late polyhedron (PH) promoter [3, 4].
Preparation of multigene expression constructs is a multistep
process with a first round consisting in the assembly of single or
dual expression cassettes. In the absence of prior knowledge about
the proteins of interest, experiments with viruses containing the
single or dual expression cassettes will provide valuable information
on expression level/solubility of individual subunits; moreover,
different affinity tags can be tested. At this stage, coinfection experiments deliver first insights into the protein–protein interaction
network and the architecture of the complex.
In the second step, expression cassettes are combined to generate multigene transfer vectors and produce the corresponding baculoviruses (Fig 1a). This can be achieved by using a combination of
restriction-ligation and in vitro Cre-mediated plasmid fusion [5–7]
as well as with restriction-free technologies such as In-Fusion [8] or
USER (Uracil-Specific Excision Reagent) cloning [9] (Fig. 1b).
We have previously described optimized protocols to generate,
evaluate, and amplify recombinant viruses, as well as to perform
expression screening and large-scale production of difficult-toexpress proteins and complexes [10, 11]. In this chapter, we focus
on the assembly of multigene expression constructs and detail
applications of homology-based cloning techniques (sequence
and ligation–independent cloning (SLIC) and similar commercially
available technologies such as In-Fusion, Gibson, or NEBuilder) to
assemble pairs of promoters to create dual expression plasmids. We
also provide a simple protocol for the modification of existing
constructs based on restriction-free (RF) cloning approach. These
protocols were implemented in our laboratory to generate constructs for the reconstitution and characterization of human multiprotein complexes that include the CAK and pTefb kinase
complexes as well as the ten subunit transcription/DNA repair
factor TFIIH (see for example [12–14]).
18
Paola Rossolillo et al.
foreign to the host cell.
With the baculovirus insect cell expression system, protein
targets can be expressed from multiple viruses carrying a single
foreign gene each, from a single baculovirus carrying multiple
foreign genes, or from a combination of the two approaches. For
routine production of complex systems, it is now widely accepted
that the use of multigene expression constructs is extremely efficient. Progress in our understanding of the baculovirus biology
together with the development of new tools for DNA manipulation
have allowed to streamline protocols for the generation of viruses,
expression screening, and large-scale productions (see refs. 1, 2 for
recent reviews). A major breakthrough was accomplished through
the development of the Multibac technology, which directs
co-expression of multiple genes from a single virus under the
control of multiple copies of the late p10 promoter and the very
late polyhedron (PH) promoter [3, 4].
Preparation of multigene expression constructs is a multistep
process with a first round consisting in the assembly of single or
dual expression cassettes. In the absence of prior knowledge about
the proteins of interest, experiments with viruses containing the
single or dual expression cassettes will provide valuable information
on expression level/solubility of individual subunits; moreover,
different affinity tags can be tested. At this stage, coinfection experiments deliver first insights into the protein–protein interaction
network and the architecture of the complex.
In the second step, expression cassettes are combined to generate multigene transfer vectors and produce the corresponding baculoviruses (Fig 1a). This can be achieved by using a combination of
restriction-ligation and in vitro Cre-mediated plasmid fusion [5–7]
as well as with restriction-free technologies such as In-Fusion [8] or
USER (Uracil-Specific Excision Reagent) cloning [9] (Fig. 1b).
We have previously described optimized protocols to generate,
evaluate, and amplify recombinant viruses, as well as to perform
expression screening and large-scale production of difficult-toexpress proteins and complexes [10, 11]. In this chapter, we focus
on the assembly of multigene expression constructs and detail
applications of homology-based cloning techniques (sequence
and ligation–independent cloning (SLIC) and similar commercially
available technologies such as In-Fusion, Gibson, or NEBuilder) to
assemble pairs of promoters to create dual expression plasmids. We
also provide a simple protocol for the modification of existing
constructs based on restriction-free (RF) cloning approach. These
protocols were implemented in our laboratory to generate constructs for the reconstitution and characterization of human multiprotein complexes that include the CAK and pTefb kinase
complexes as well as the ten subunit transcription/DNA repair
factor TFIIH (see for example [12–14]).
18
Paola Rossolillo et al.