Insect Cells

Insect cells obviously resemble mammalian cells in many aspects and are therefore attractive for recombinant protein production. Development of baculovirus vectors for heterologous gene expression has been very successful.36 Generally, the strong polyhedrin promoter from Autographa californica has been applied for expression in several insect cell lines from organisms such as Spodoptera frugiperda (Sf9 and Sf21 cells) and Trichoplusia ni (Tni and High FiveTM cells).37 Insect cells possess many of the mammalian post-translational modifications, although certain differences between mammalian and insect cell pathways exist.

For example, N-glycosylation in insect cells is simpler and of high mannose type.38 In addition to insect cells, baculovirus is also capable of infecting mammalian cells. In this context, replacement of the polyhedrin promoter with a CMV promoter allowed expression of recombinant proteins from baculovirus vectors also in mammalian cells.39 However, one drawback of this approach was the requirement for very high virus concentrations — MOI (multiplicity of infection) levels in the range of 500 — which made large-scale production unfeasible.

Baculovirus vectors have been widely used for GPCR expression in insect cells resulting in high expression levels with Bmax values in the range of 20 to 80 pmol/mg.40 They include a large number of different GPCRs such as adrenergic, dopamine, muscarinic, opioid, tachykinin, and serotonin receptors. In attempts to further improve folding and transport to the plasma membrane and thus enhance expression levels, various signal sequences were fused to the N-termini of GPCRs. The mellitin sequence from Apis melifera improved the expression level of the human dopamine D2S receptor approximately two-fold.41 Similar observations were made for the introduction of the influenza virus hemagglutinin signal sequence in front of the human p2 AR.42

The signal sequence from the baculovirus gp64 protein enhanced the expression levels of the human m opioid receptor43 and of various serotonin receptors.44 Another approach is the engineering of the host insect cells to facilitate the folding process by co-expression of chaperon proteins such as BiP (immunoglobulin heavy chain binding protein) in the endoplasmic reticulum (ER) and calnexin residing in ER membranes.45 Appropriate attention has also been paid to cell culture conditions. When Sf9 cells were grown in serum-free medium, yields for the human m opioid receptor doubled.43

The locations of purification tags play crucial roles in relation to expression levels. Typically, engineering of His6 tags at the N-termini of opioid receptors reduced the expression levels substantially, whereas the effect was less dramatic when introduced at the C-terminus.46 Deletions in gene constructs were also shown to affect expression levels. A truncated turkey p-adrenergic receptor with deletions in both the N- and C-terminals to remove all N-glycosylation sites and an additional point mutation Cys116Leu improved yields more than five-fold in Tni cells.47 This led to record high expression levels with Bmax values of 360 pmol/mg that from large-scale production provided 10 mg of p-AR for crystallization.

As an alternative to using baculovirus for GPCR expression in insect cells, stable inducible expression has been developed under the control of the metallothio-nein promoter in Drosophila Schneider-2 cells. The expression levels of human opioid receptors have been relatively low, in the range of 20,000 to 30,000 receptors/cell.48 However, fusion of GFP to the C-terminus allowed quantitative fluorescence intensity analysis, suggesting that the total receptor level was eight-fold higher than the diprenorphine binding data indicated. This may be due to problems with folding and transport of recombinant receptors. GFP quantification and monitoring could prove a versatile tool to develop further technology to improve expression levels in insect cells.

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