Recombinant Protein Expression in Insect Cells
In parallel with the development of plasmid systems, another pivotal element to facilitate the production of the most biologically relevant recombinant proteins is the selection of expression host. While E.coli is often considered as a major work-horse for recombinant protein expression, it has limited capability for protein folding and also lacks the machinery to add suitable post-translational modifications onto target proteins. Advanced eukaryote cells, such as yeast, insect cells, and mammalian cells (HEK293 and CHO) have been developed. However, every system has its advantages and limitations (Table 1).
Table 1: commonly used host cells for recombinant protein expression
Insect cells require an intermediate, baculovirus, for protein expression. Baculoviruses are a diverse group of DNA viruses that are capable to infect various (>600) insect cells. They serve as a shuttle for the introduction of the target gene into a given host cell. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best characterized and utilized baculovirus for this purpose. A flow chart of the process is summarized in Figure 1. Briefly, a gene encoding the protein-of-interest is inserted into a primary vector, which is subsequently cloned into a secondary vector called "Bacmid". The Bacmid is transferred into a bacteria strain (commonly E.coli) for preliminary virus production and assembly to acquire generation 1 baculovirus (P1). The P1 virus is amplified in an insect cell (e.g. sf9) to reach the suitable titer (P2) and the P2 virus is then used to infect the same or a different insect cell line (e.g. High-five) for protein expression.
Figure 1: flowchart for recombinant protein expression in insect cells.
Application notes and case studies
Insect cells are versatile expression hosts for a range of recombinant proteins. The strong folding capability and relatively high culture density make them excellent choices for the expression of complicated intracellular protein and virus proteins. In 2007, Cervarix, an HPV vaccine produced by an insect cell line in the format of virus-like particles (VLPs) was approved for human use. Besides potentials in therapeutic and vaccine development, highly active proteins produced in insect cells are widely used in a variety of disciplines in biophysics and biochemistry for structure elucidation, drug design, assay establishment, and diagnostic reagent development.... Sino Biological uses insect cells extensively for recombinant protein product development and contracted research projects. Here, we would like to present a few application notes and case studies to demonstrate the power and versatility of the insect cell expression system.
- Cell line: we routinely use sf9 as virus amplifier and High-five as production cell line. We observe a relatively higher protein expression when using High-five in general
- Cell culture: Sino Biological has developed a proprietary culture medium, designated as SCD6SF medium (currently available only within mainland China), for insect cell culture. We maintain our culture at ~ 6x106 cells/mL for small-scale protein expressions.
- Signal peptide: insect cells are quite adaptable for signal peptides from different species. We find signal peptides from mammals and viruses can normally be used to guide protein secretion in insect cells while signal peptides from other species, e.g. plants, bacteria, and aquatic creatures in general, are less well accommodated. In these cases, insect secretion signal peptides, such as honeybee melittin signal peptide, gp64, and gp67..., shall be considered as an alternative.
- Extraction protocol: for intracellular proteins, methods for cell lysis and protein extraction play vital roles to stabilize target protein for subsequent purification. For any given protein, we recommend a pilot study to establish the most suitable extraction protocol, including but not limited to, cell lysate duration and method, lysate buffer recipe, extraction buffer pH, buffer component (PBS, Tris, HEPES...), salt concentration, type and concentration of detergent.... A small-scale purification test by the designated method is also recommended to assess the yield and purity of the final protein product.
- Protein construct
Certain structural features on a target protein (regions of high hydrophobicity, high disorderness, and repetitive amino acid motifs...) might cause instability in recombinant protein expression. As long as such regions are not directly involved in protein function, their removal may help improve protein expression (Figure 2)
Figure 2: removal of a hydrophobic region in the protein sequence to enhance protein expression in insect cells.
Proteins are sensitive to their surrounding chemical environment. Changes in pH, ionic strength, and oxidative status... would impact protein stability. During purification, sometimes additives are also needed to stabilize target protein or facilitate tag exposure. As shown in Figure 3, the target protein (a single-pass transmembrane protein) was poorly extracted using detergent formula 1, presumably due to the inadequate exposure of the His-tag. Revision of the detergent formula enhanced protein extraction while during the final polishing step, another detergent (DDM) was used to replace detergent formula 2 and stabilize the final protein product.
Figure 3: buffer optimization during protein purification to enhance protein recovery in insect cells.
Recombinant proteins are fundamental to the current biologics development landscape. As an excellent choice of expression host, insect cells enable correct protein folding, PTM, and they are suitable for high-density cell culture and produce both secreted and intracellular proteins across various species. Certain technical know-how is required for protein design and a systematic optimization approach is essential for obtaining high-quality recombinant proteins via insect cells.