Baculovirus expression: tackling the complexity challenge

Abstract

Most essential functions in eukaryotic cells are catalyzed by complex molecular machines built of many subunits. To fully understand their biological function in health and disease, it is imperative to study these machines in their entirety. The provision of many essential multiprotein complexes of higher eukaryotes including humans, can be a considerable challenge, as low abundance and heterogeneity often rule out their extraction from native source material. The baculovirus expression vector system (BEVS), specifically tailored for multiprotein complex production, has proven itself to be uniquely suited for overcoming this impeding bottleneck. Here we highlight recent major achievements in multiprotein complex structure research that were catalyzed by this versatile recombinant complex expression tool.

Graphical abstract

Figure 1

S. cerevisae Mediator Complex. (a) Subunit organization of Mediator. Head module subunits are colored in blue; those of the Middle module are in green and those of the Tail module are in ochre. (b) Genes encoding the Mediator Head module were assembled from multiple DNA progenitors (left) into a single multigene expression construct (right, top) for insertion by Tn7 transposition (Tn7L, Tn7R) into the MultiBac baculovirus to produce the Head module in infected insect cells (right, bottom). (c) X-ray crystal structure of the recombinant Mediator Head module showing Med17 (blue), Med11 (purple), Med22 (green), Med6 (yellow), Med8 (red), Med18 (cyan), and Med 20 (orange). The Head consists of three distinct domains, fixed jaw, movable jaw and neck (left). The neck domain is arranged in a novel multihelical bundle (right) [30^••].

Figure 2

Structures of the APC/C and MCC. 3-D EM reconstructions of (a) recombinant S. cerevisiae APC/C and (b) endogenous S. cerevisiae APC/C. (c) Localisation of the Cdc16 dimer in Cdc16-assigned density (mesh). (d) Pseudo atomic structure of S. cerevisiae APC/C, adapted from [34^••]. (e) Endogenous human APC/C [51^•]. (f) Recombinant human APC/C [35]. (g) Structure of the S. pombe mitotic checkpoint complex [50^•].

Figure 3

Human general transcription factor TFIID core complex. (a) Generally applicable polyprotein strategy for balancing the production levels of multiprotein complex subunits. Genes of interest (GOI), a protease encoding gene (TEV NIa) and a fluorescent marker (CFP) are present in a single ORF under control of a strong baculoviral promoter (polh) and flanked by a poly A signal (black square) on a MultiBac plasmid (cf. Figure 1B), spaced apart by the specific cleavage sequence of TEV NIa (tcs) [1•, 58]. (b) Polyprotein expression and purification of human core-TFIID. Negative stain EM and 2-D classification (bottom, right) were used to optimize purification until high-quality sample was obtained as demonstrated by SDS-PAGE (bottom, left). (c) TFIID core complex structure at nanometer resolution, determined by hybrid methods [57^••].