BIOTECHNOLOGY
Cellular Protein Synthesis P
Exploring recombinant protein expression using a baculovirus-insect cell system
roteins are essential for various cellular functions, making them a focal point of scientifi c inquiry across
disciplines. Understanding the structure and function of proteins is crucial for unlocking the ‘mysteries of life’, driving research eff orts across several scientifi c fi elds. However, obtaining proteins for study can be challenging due to their complexity. Recombinant protein expression
is a method that can help solve this problem since it produces proteins by cloning the gene of interest into an expression vector and introducing it into a host cell. The selection of the optimal host cell system is critical for successful protein expression. One such system is the Baculovirus-insect Cell System (BEVS), which utilises baculoviruses to deliver target genes into insect cells for protein production.
THE BEVS PROCESS In the BEVS process, a gene encoding the protein-of-interest is inserted into a primary vector and then cloned into a secondary vector known as Bacmid. This Bacmid is transferred into a bacterial strain for preliminary virus production, resulting in the generation of baculovirus (Figure 1). The baculovirus is then amplifi ed in insect cells and used to infect insect cell
Figure 1: Flowchart for recombinant protein expression in insect cells
lines for protein expression, off ering a versatile platform for producing various proteins.
APPLICATION OF BEVS Insect cells are often used to
produce large molecular weight (MW) proteins (MW> 150 kDa) because of their superior folding and post- translational modifi cation capabilities. One major downside of this endeavour, however, is the structural complexities of such proteins, which often results in relatively low yields (Fig. 2, left). One 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
Figure 2: Expression of full-length (left), truncated (middle), and methyltransferase plus ketoreductase domain fusion (right) constructs of human FASN. The full- length protein was prepared as reported by Hardwicke et al.(2014,doi:10.1038/ nchembio.1603), whereas the domain fusion construct was prepared as reported by Lu et al.(2018, doi: 10.1016/
j.bmcl.2018.05.014)
not feasible because of low protein yield and heavy degradation (Fig. 2, middle). By extrapolating and fusing the sequences encoding the methyltransferase and ketoreductase domain with a linker, Sino Biological successfully obtained a high-yield construct (Fig. 2, right). The Elute 1 and 2 of this construct were pooled and further purifi ed to yield a fi nal fusion protein with >90% purity. It should be noted that some proteins require certain oligomeric formations to be functional. For example, the hemagglutinin proteins of infl uenza virus and the spike protein of SARS-CoV-2 exhibit a trimeric format, whereas the human prolyl endopeptidase, FAP, is an intrinsic dimer.
CONCLUSION 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 modifi cation, and are suitable for high-density cell culture. In addition, they can produce both secreted and intracellular proteins of various species. ■
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