Baculovirus Expression Strategies for Protein Complex Production

Abstract

Protein complexes often contain multiple subunits, and it is these complexes that are at the core of biological function in the cell. Inherently low quantities of protein complexes in eukaryotes can prohibit the extraction of these essential cellular machines from native source material, posing a considerable challenge for functional analysis at the molecular level. The baculovirus expression vector system (BEVS) is particularly useful for recombinantly producing eukaryotic proteins in the quality and quantity required for deciphering their structure and function. Recent efforts have focused on improving reagents and streamlining protocols to routinely express protein complexes by recombinant baculoviruses. The challenge of integration of baculovirus expression in high‐throughput production pipelines for proteins and their complexes is being addressed.

Key Concepts:

  • Protein complexes are a cornerstone of biological function in the cell.

  • Recombinant production is essential to study many multiprotein complexes.

  • Ongoing development of expression systems for producing multiprotein complexes.

  • BEVS as a method of choice for large‐scale production of complexes requires integration of all genes in a single multigene baculovirus rather than coinfecting insect cell cultures with many baculoviruses.

  • Creation of new reagents and streamlined protocols to address the challenge of generating multigene baculoviruses for routine laboratory use.

  • Engineering baculoviruses and new host cell lines to further enhance protein production by BEVS.

  • Multigene vector generation incorporated into automation pipelines to increase the throughput of protein complex expression by BEVS.

Keywords: multiprotein complexes; interactome; eukaryotic expression; BEVS; MultiBac system; BAC; insect cell culture; automation; structural biology

Figure 1.

Recombinant protein production by BEVS. (a) Schematic drawing of baculovirus virion resembling a stick (‘baculum’) in appearance. The large double‐stranded genome (dark spiral, centre) resides in a flexible envelope, which forms a round protrusion towards one end. Large heterologous DNA insertions into the baculoviral genome, encoding for single proteins or protein complexes of interest, can be accommodated due to this flexible architecture, resulting in fully functional virus that is infectious and can produce heterologous gene product. The envelope of the virion is composed of lipid raft acquired from the host insect cell during virus production. Baculovirus‐encoded glycoproteins (black rods) form trimers on the surface of the virion (Courtesy of K. Airenne). (b) The baculovirus infectious cycle as exploited in the laboratory for protein production is shown. A variety of methods exist to introduce heterologous genes encoding for recombinant proteins (denoted Gene) into the baculoviral genome (bottom left). The resulting recombinant baculovirus (top left) is used to transfect insect cell cultures in monolayer or suspension, aided by lipid transfection reagent. The baculovirus genome is transported to the nucleus of the infected insect cells. Initially, the host cell machinery is used to initiate the infectious cycle of the baculovirus. Virions are produced at maximal rate in the first 20–30 h and are budded off the host cell (top right). Small but detectable amounts of the heterologous protein of interest are already produced, usually from promoters that attain peak activity only at the very late stage of viral infection. Heterologous protein production is maximal at around 72 h after infection (bottom right). Recombinant proteins are often authentically processed and posttranslationally modified in insect cells and targeted to the appropriate cell compartment. Thus, recombinant proteins can be purified from the cytosol or the nucleus, or they can be secreted into the cell culture medium from where they can be recovered. During the 30‐year span that BEVS has been available, thousands of recombinant proteins have been successfully produced for downstream applications ranging from basic research to medical applications including therapeutic interventions and vaccination. The recombinant gene is in dark blue, the baculovirus genome orange and expressed proteins are shaded in blue.

Figure 2.

Heterologous gene insertion into baculovirus. (a) Purified baculoviral genomic DNA can be mixed with a transfer plasmid containing the heterologous gene of interest (denoted Gene) and a lipid transfection reagent, to transfect insect cells (I). The gene in the transfer plasmid is flanked by DNA regions (green and white boxes) that are also present on the baculovirus. Homologous recombination occurs in insect cells that receive both DNAs (marked with X), giving rise to a recombinant baculovirus in which the polh gene is replaced with the gene of interest. This original procedure is very inefficient and as a result has become outdated. Digestion of the baculovirus genome by Bsu36I restriction enzyme at a unique site upstream of the polh gene gives rise to linearised baculoviral genomic DNA, which cannot replicate, thereby improving the efficiency of recombinant virus generation significantly (II). A further improvement of this approach uses baculoviral DNA where the entire polh locus as well as parts of the adjacent baculoviral ORFs (ORF603, ORF1629) have been excised (III). The transfer plasmid used in conjunction with this baculoviral DNA contains DNA sequences corresponding to the full‐length ORFs, flanking the gene of interest. Homologous recombination in insect cells introduces the gene of interest and simultaneously regenerates the complete ORFs. ORF1629 is an essential viral gene; therefore, only productively recombined recombinant baculoviral DNA can undergo the infectious cycle of the virus. (b) The BAC/Tn7 approach for gene insertion is shown. The baculovirus genome exists as a bacterial artificial chromosome in E. coli cells expressing the Tn7 transposon complex (right). The gene of interest is introduced by transforming the plasmid into these E. coli cells. This transfer plasmid (left) contains short DNA sequences called Tn7R and Tn7L (shown as black triangles) flanking the gene of interest and a resistance marker (box coloured red). Upon transformation, the Tn7 transposon integrates the DNA in between the Tn7L and Tn7R sequences into a Tn7 attachment site (black box) embedded in a LacZα gene (light blue). Positive integrants are identified by blue/white screening and by selecting for the resistance marker cointegrated into the gene. Recombinant baculoviral DNA is prepared from E. coli minicultures for transfection of insect cells. (c) A method combining both approaches is shown. Here, the baculoviral DNA contains an F replicon (marked F) in between ORF603 and a truncated ORF1629Δ. The F replicon allows propagation of this baculovirus as a BAC in bacterial cells for mass production. Integration of the gene of interest is achieved by cotransfection with a transfer plasmid containing the gene flanked by DNA sequences corresponding to the full‐length ORFs. Homologous recombination yields a recombinant baculovirus containing the gene of interest, which is replication competent as the essential ORF1629 is fully restored. This method is arguably the most efficient approach currently for integrating heterologous genes into baculovirus.

Figure 3.

Strategies for multiprotein complex production by BEVS. (a) Coinfection with multiple viruses is illustrated. Genes (denoted a, b, c and d) encoding for subunits of a given protein complex are inserted each into one baculovirus. The resulting baculoviruses are produced and amplified separately, and their viral titres determined. The baculoviruses are mixed in a defined ratio according to their viral titres and individual protein production performance, and the cocktail is used to infect insect cell cultures. Cells in the culture which receive all viruses in an appropriate ratio will produce the desired protein complex (subunits marked A, B, C and D). Misbalanced virus ratios, in particular if titre determination is inaccurate, can lead to the production of incomplete complexes lacking individual subunits when applying this method. Furthermore, it is not trivial to ascertain that all cells receive all viruses. Likewise, the ratio of viruses received by the individual cells in a culture can be subject to considerable variation. (b) Multiprotein expression from a single multigene virus is depicted, using the MultiBac system as an example. Genes are inserted into an array of small plasmid molecules that are fused by Cre‐LoxP reaction to a single multigene transfer plasmid containing the Tn7R and Tn7L sites. Encoding genes (denoted a, b, c, d and e) are integrated into the MultiBac baculovirus by Tn7 transposition in bacteria harbouring the viral genome as a BAC, and expression the Tn7 transposon. The resulting composite bacmid contains all genes encoding for the complex, and in addition, a yellow fluorescent protein (YFP) marker for tracking virus performance and protein production by measuring the YFP fluorescence signal. Every cell in the insect cell culture that is infected by the resulting virus will express all components of the multiprotein complex of interest (labelled A, B, C, D and E) at the ratio dictated by the expression characteristics of the individual proteins. In addition, YFP is produced, which is separated from the multiprotein complex during purification. All encoding genes are expressed from two loci in the MultiBac virus. (c) An alternative method for the expression of protein complexes by iterative modification of baculoviral genomic bacmid DNA is shown. A linear DNA molecule is generated, which contains a gene encoding for a subunit of the complex (marked a). In addition, the linear fragment contains promoter and terminator elements flanking the gene, a selection marker in between two LoxP sites (box shaded in dark gre), and a short homology region from a specific locus on the baculovirus at either end of the DNA (black and white boxes, respectively). This DNA fragment is transformed into E. coli cells containing a baculovirus genome as a BAC. In addition, these cells express the components of the lambda red recombinase (λRED) and contain a plasmid from which Cre recombinase production can be induced. The DNA fragment containing the gene of interest is integrated into the locus specified by the short homology regions. Recombinant bacmid is identified by selecting for the resistance marker that is cointegrated. Next, production of Cre recombinase is induced, catalysing excision of the selection marker by rejoining the two flanking LoxP sites. The process can now be repeated iteratively (shown here for two further genes, b and c). The resulting multigene baculovirus contains heterologous genes spaced apart by viral sequences, which may enhance the stability of the recombinant baculovirus. Protein complex (subunits denoted A, B and C) is produced by infecting insect cell cultures with this single multigene virus.

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Sumitra Vijayachandran, Lakshmi, Viola, Cristina, and Berger, Imre(Apr 2011) Baculovirus Expression Strategies for Protein Complex Production. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0023180]