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Creative Biolabs offers advanced Mempro™ membrane protein production based on insect cells system. The baculoviruses are widely used for eukaryotic membrane protein production in cultured insect cells and insect larvae.
Mempro™ protein production in insect cells system is more compatible with eukaryotic proteins due to similar codon usage rules, providing better expression levels and fewer truncated proteins than in E. coli system. This system allows for post-translational modifications, moreover, which are easier and cheaper to handle than adherent cells like HEK 293, COS or CHO cells, especially for scale-up. Briefly, the baculovirus system relies on the infection of insect cell lines (usually Sf9 or Sf21) by recombinant viruses encoding the target membrane proteins. Notably, Sf9, a clonal isolate of the Spodoptera frugiperda cell line IPLB-Sf21-AE, is the most widely used for baculovirus-based membrane protein production. Sf9 was originally established from ovarian tissue of the fall armyworm.
Figure 1. Heterologous protein expression in insect cells. (Trends in Mol. Med., 2010)
Wild-type baculoviruses exhibit both lytic and occluded life cycles that develop independently throughout the three phases of virus replication: 1). Early phase, is known as the virus synthesis phase. 2). Late phase, is known as viral structural phase, and starts to produce budded virus (BV). 3). Very late phase, is known as the viral occlusion protein phase, the polyhedrin and p10 genes are expressed, and occluded virus (OV) are formed. Two of the most common baculoviruses used in heterologous protein expression are Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Bombyx mori (silkworm) nuclear polyhedrosis virus (BmNPV).
The major feature of the recombinant baculovirus in vitro infection is that the naturally occurring polyhedrin gene within the wild-type baculovirus genome is replaced with a recombinant gene or cDNA, which is commonly under the control of polyhedrin and p10 promoters. In the late phase of in vitro infection, the virions are assembled and budded recombinant virions are released. However, during the very late phase of infection, the inserted heterologous genes are placed under the transcriptional control of the strong AcNPV polyhedrin promoter. Thus, recombinant membrane proteins are expressed in place of the naturally occurring polyhedrin protein. Usually, the recombinant proteins are processed, modified, and targeted to the appropriate cellular locations.
The advantages of Mempro™ protein production in insect cells system based on baculovirus are described as below:
C. Trometer, et al. (2010). Mammalian membrane protein expression in baculovirus-infected insect cells. Methods Mol. Biol., 601: 105-117.
F. Bernaudat, et al. (2011). Heterologous expression of membrane proteins: choosing the appropriate host. PLoS One, 6(12): e29191.
F. Krammer, et al. (2010). Alternative influenza vaccines made by insect cells. Trends in Mol. Med., 16(7): 313-320.
N. J. Hu, et al. (2015). GFP-based expression screening of membrane proteins in insect cells using the baculovirus system. Methods Mol. Biol. 1261: 197-209.
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