Recombinant Protein Expression with a Baculovirus–Insect Cell System

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Proteins create cellular matrices, catalyze biochemical reactions, and form signaling pathways to respond to external stimuli. Studies of protein structure and function increase researchers’ understanding about the foundations of life. However, because most proteins are difficult to obtain commercially, it is important to establish sources that can provide researchers with plentiful supplies of proteins. In recombinant protein expression, a gene encoding a protein of interest is cloned into an expression vector (usually a plasmid) and transferred into a host cell for protein production by harnessing the cell’s intrinsic protein synthesis machinery. Several host-cell systems have been established for recombinant protein production. Selection of the optimal host for a given protein is a major factor for successful expression. Table 1 lists the advantages and limitations of commonly used expression hosts.

Table 1: Commonly used host cells for recombinant protein expression.

Baculovirus Expression Vector System (BEVS)
To deliver a target gene into cells and achieve protein expression, insect cells require baculovirus as an intermediate. Baculoviruses represent a diverse group of DNA viruses that can infect >600 different types of insect cells. Baculoviruses serve as a shuttle to introduce a target gene into a host cell. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-characterized baculovirus for that purpose, and it is widely used for insect-cell–mediated protein expression.

Figure 1 shows a flow chart of a recombinant protein expression process from insect cells. A gene encoding a protein of interest is inserted into a primary vector, which subsequently is cloned into a secondary vector known as a Bacmid. That is transferred into a bacteria strain (usually Escherichia coli) for preliminary virus production and assembly to obtain generation 1 baculovirus (P1). The P1 virus is amplified in an insect cell (e.g., Sf9 cells) to achieve a suitable titer (P2), which then is used to infect the same or a different insect cell line (e.g., High Five cells) for protein expression. This Bac-to-Bac system (originally developed by Invitrogen, now part of Thermo Fisher) has been adapted for the expression of several proteins as secreted, intracellular, or membrane-bound molecules. Many other methods for baculovirus generation are commercially available.

Figure 1: Flowchart for recombinant protein expression from insect cells.

Application of BEVS
Insect cells are versatile expression hosts for different recombinant proteins. They are excellent choices for the expression of complicated intracellular and viral proteins because of their robust protein-folding capability and relatively high culture densities. In 2007, Cervarix (GlaxoSmithKline), a human papilloma virus vaccine produced using an insect cell line in the format of virus-like particles was approved for human use. Highly active proteins produced in insect cells are used in several activities, including structure elucidation, drug design, assay establishment, and diagnostic reagent development. To obtain fully functional recombinant proteins, a systematic design is required for each step from construct design and culture optimization to protein purification and protein formulation. Below, we describe two cases to demonstrate key features of recombinant protein expression using BEVS.

Core Region Fusion: Insect cells often are used to produce large–molecular-weight (MW) proteins (>150 kDa) because of the cells’ superior folding and posttranslational modification capabilities. One major downside of this application, however, is that the proteins have structural complexities. That often results in relatively low yields (Figure 2, left). An alternative approach is to express a domain of interest rather than the entire protein. However, in the case presented here, the direct expression of two enzyme domains of human fatty acid synthase (FASN) was not feasible because of low protein yield and heavy degradation (Figure 2, middle). By extrapolating and fusing the sequences encoding the methyltransferase and ketoreductase domain with a linker, we obtained a high-yield construct (Figure 2, right). Elute 1 and 2 of that construct were pooled and further purified to yield a final fusion protein with >90% purity.

Figure 2: Expression of full-length (), truncated (), and methyltransferase plus ketoreductase domain fusion () constructs of human fatty acid synthase; the fulllength protein was prepared as reported by Hardwicke et al. (1). (FT = flowthrough, AA = amino acid).

Obtaining Protein with the Correct Oligomeric Status: Some proteins require certain oligomeric formations to be functional. For example, the hemagglutinin proteins of influenza virus and the spike protein of SARS-CoV-2 exhibit a trimeric format, whereas human prolyl endopeptidase fibroblast activation protein (FAP) is an intrinsic dimer. Caution should be exercised during purification steps to track the protein fractions with the correct oligomeric status and ensure proper protein activity. In this study, a histidine-tagged protein was expressed using BEVS, and the nickel-affinity eluate contained a mixture of its monomer and dimer. Because the protein is active as a dimer, a second gel-filtration purification step was required to enrich the protein dimer. The dimeric conformation of the final product was confirmed using size-exclusion chromatography–high-performance liquid chromatography (SEC-HPLC), and the protein exhibited reproducible enzymatic activity between two different batches using the established method (Figure 3).

Figure 3: Expression and purification of an active protein dimer; the Ni-affinity Eluate 2 was subjected to gel-filtration chromatography to enrich for the protein dimer. The dimeric status of the protein was assessed by size-exclustion chromatography (SEC) and the activity was determined using the corresponding enzymatic assay. (E1, E2 = Elute 1 and 2, MW = molecular weight, SDS-PAGE = Sodium dodecyl-sulfate–polyacrylamide gel electrophoresis, FT = flowthrough).

Selecting Insect Host Cells
Recombinant proteins are fundamental to the study and development of biologics. Insect cells are a superior choice as an expression host because they enable correct protein folding and posttranslational modification, and they are suitable for high-density cell culture. They can produce both secreted and intracellular proteins of various species. A systematic design and optimization approach is essential to obtaining high-quality recombinant proteins from insect cells.

1 Hardwicke MA, et al. A Human Fatty Acid Synthase Inhibitor Binds β-Ketoacyl Reductase in the Keto-Substrate Site. Nat. Chem. Bio. 10, 2014: 774–779;

Dr. Yuning Chen is senior R&D director at Sino Biological;