Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering

Abstract

Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach.

Keywords: BEVS; MultiBac; OmniBac; baculovirus; comparative genome analysis; multiprotein complexes; recombinant protein production.

Figure 1. OmniBac. (A) Synthetic donor and acceptor plasmids are shown schematically. Donors pIDC, pIDK and pIDS are from the original MultiBac suite. Novel acceptor plasmids pOmniBac1, pOmniBac2 and pOmni-PBac are shown. Cm stands for chloramphenicol, Kn for kanamycin, Sp for spectinomycin, Gm for gentamycin. Viral very late promoters polh and p10, and SV40 and HSVtk polyadenylation signals are indicated. Donors contain a conditional origin of replication derived from the R6Kγ phage. A multiplication module comprising a homing endonuclease site and a complementary BstXI site is shown as boxes in light blue, flanking the expression cassettes. MCS1 and MCS2 denote multiple cloning sites.^, Acceptors contain a regular ColE1 origin of replication, the DNA sequence elements for Tn7 transposition (Tn7L, Tn7R) and in addition the lef2/603 and Ori1629 homology regions (gray boxes). All plasmids have a LoxP sequence for plasmid fusion mediated by Cre recombinase. pOmni-PBac contains the polyprotein expression cassette for multiprotein complex expression including a gene encoding for tobacco etch virus Nia protease (TEV) followed by a fluorescent marker, cyan fluorescent protein (CFP), spaced by a TEV protease cleavage site (tcs). Polyprotein constructs are generated in pOmni-PBac as described previously for our pPBac design. (B) OmniBac acceptors and donors are concatenated by Cre-LoxP fusion in a combinatorial fashion.^, For matter of clarity, only two donors are shown reacting with one acceptor, each containing one heterolgous gene (white arrow). Unfused donors act as suicide plasmids upon transformation due to their conditional origin. AD and ADD fusions, in contrast, are efficiently propagated based on the regular origin in the acceptor used. Plasmid combinations are selected based on unique resistance marker combinations. (C) OmniBac acceptors contain all elements required for generating recombinant baculoviruses for expression by using the homologous recombination method (left) or, alternatively, the BAC/Tn7 entry method (right). (D) An OmniBac plasmid containing two genes encoding for CFP (top) was used to test homologous recombination entry (BacVector3000, FlashBac) as well as the BAC/Tn7 entry (Bac-to-Bac, MultiBac). Quantification of fluorescence on an arbitrary scale calibrated to a yellow fluorescent protein standard (YFP) and 1 million infected cells each exhibited virtually identical heterologous expression levels (two clones each were tested). (E) Connexin (left) and Drebrin (right) were expressed using OmniBac and purified to homogeneity. Connexin migrates as an oligomer in SDS-PAGE.

Figure 2. Blueprint of the baculovirus genome. The annotated genome of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) is shown in a schematic representation (top). Genes are scored (bottom) on a scale from essential genes that are conserved (no deletion category, shades of green color to black) to non-essential genes (shown to be possibly deleteable, shades of red color). The classification applied is detailed in the legend (bottom). The annotated genome map was generated by a self-developed Perl program. Essential and non-essential genes are not randomly distributed but cluster in the genome. The upper semicircle is composed to 56% of the essential genes (and 44% of non-essential genes), the lower, more conserved semicircle is composed to 71% of essential genes (and 29% of non-essential genes).