Recombinant protein expression

Proteins are the key products of the “central dogma of biology” and they are basic the building blocks of life as we know it. Proteins perform a variety of tasks from creating cellular matrixes, catalysing biochemical reactions, and forming multiple signalling pathways to respond to external stimuli. The studies of the structure and function of proteins hold the key to understanding the “meaning of life” and they are thus pursued by scientists across multiple disciplines. However, due to the scarcity of most proteins of interest, it is important to secure a source that could potentially provide researchers with an “unlimited supply” of proteins. Thus enters the concept of recombinant protein expression, where genes encoding target proteins-of-interests are cloned into expression vectors (usually as plasmids) and transferred into various host cells for protein production by harnessing the intrinsic protein generation machineries within the hosts. Several host cell systems have been established for recombinant protein production and the selection of the most suitable host for any given protein is a major factor that contributes to its successful expression. The advantages and limitations of commonly used expression hosts are summarised in Table 1.

Insect cells require an intermediate, baculovirus, to deliver the target gene into the cells and achieve 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 baculovirus for this purpose and it is widely used in insect cell mediate protein expressions. A flow chart of the process is summarised 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. This “Bac-to-Bac” system has been well adapted in the expression of a wide variety of proteins as secreted, intracellular, as well as membrane-bound formats. It is worth noting that there are other commercially available methods for baculovirus generation.

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. Highly active proteins produced in insect cells are also widely used in a variety of disciplines in biophysics and biochemistry for structure elucidation, drug design, assay establishment, and diagnostic reagent development. To obtain fully functional recombinant proteins, a systematic design is required to plan each step from construct design, culture optimisation, all the way to method of purification as well as protein formulation. Two cases are selected and presented here to demonstrate key points in recombinant protein expression using BEVS.

(1) Core region fusion

Insect cells are sometimes used to produce large molecular weight (MW) proteins (WM> 150 KDa) due to their superior folding and post-translational modification capabilities. One major downside of such endeavour is that due to the structural complexities of such proteins, they are often expressed at relatively low yield (Fig 2, left). One alternative approach would be to express the domains-of-interests rather than the whole protein. However, in the case presented here, the direct expression of the two enzyme domains of human fatty acid synthase (FASN) was infeasible due to low protein yield and heavy degradation (Fig 2, middle). By extrapolating and fusing the sequences encoding the methyltransferase domain and the ketoreductase domain with a linker, we successfully obtained a high-yield construct (Fig 2, right). The Elute 1 and Elute 2 of this construct were pooled and further purified to yield a final fusion protein with >90% purity. 

(2) Obtaining protein with correct oligomeric status

Some proteins require certain oligomeric formation to be functional. For instance, the hemagglutinin (HA) proteins of influenza virus and the spike protein of SARS-CoV-2 exert a trimeric format while the human Prolyl endopeptidase FAP is an intrinsic dimmer. Cautions should be taken in the purification steps to track the protein fractions with the correct oligomeric status to ensure proper protein function. A protein (His-tagged) was expressed using the BEVS and the Ni-affinity eluate was a mixture of monomer and dimmer. Since the protein is active in the dimmer formation, a second gel-filtration purification step was used to enrich the protein dimmer. The dimmeric status of the final product was confirmed and the protein showed reproducible enzymatic activity between two different batches using the established method.

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. A systematic designing and optimisation approach is essential for obtaining high-quality recombinant proteins via insect cells.