Glycoprotein expression via gene modified insect cell lines

Due to the insufficient of N-acetylglucosaminyltransferase II (GlcNAc TII) activity, it is difficult for insect cells to produce complex, terminally sialylated biantennary N-glycans, consequently cannot meet the needs of post-translational glycosylation of mammalian proteins. The difference of N-glycan processing pathway between insect cells and mammalian cells limits the development of baculovirus mediated glycoprotein expression. 

Figure 1. The process of N-glycosylation in different cells

Technology Description

Studies have shown that the N-glycan processing pathway in Lepidoptera cells can be expanded by adding mammalian genes encoding functions, which are missing or limited previously in insect cells. Based on this conclusion, many transgenic cell lines, which can express mammalian function encoding genes, have been developed in recent years.

As an expert in the field of protein expression, Creative BioMart has been able to provide non-secretory N-linked glycoprotein expression services via a novel sf9 cell line.

We used a genetically modified insect cell line that integrated with N-acetylglucosaminyltransferase II (GlcNAc TII) open reading frames (ORF) in the genome.

Features of the technology

 The stable transgenic host cell line—SfSWT-1

• Has higher levels of activitiey of GlcNAc TII, β,4-galactosyltransferase (β 4 Gal-T) and α2,6-sialyltransferase (ST6GalI) . Influence of baculovirus infection on glycosyltransferase activities is showen as Figure 2.

Figure 2. Influence of baculovirus infection on glycosyltransferase activities. The activitiey of GlcNAc-TII (A), β4Gal-T (B), and ST6GalI (C) of Sf9 (circles) or SfSWT-1 (squares) cells after infected with wild-type baculovirus.

• The cell line support normal level of baculovirus infection and it does not interrupt virus replication. Figure 3 shows the virus growth cerve after infect both sf9 and SfSWT-1.

Figure 3. Wild-type baculovirus growth cerve after infect both sf9 (circles) and SfSWT-1 (squares).

• The SfSWT-1 cell line can produce biantennary, terminally sialylated N-glycans.
• Infection with baculovirus does not shut down host gene expression at the transcriptional level of the cell line. Different from other cell lines, the activity of glycosyltransferase in SfSWT-1 cells remained at a high level at least 72 hours after infection.

It will be widely useful for the production of more authentic recombinant glycoproteins by baculovirus expression vectors. Through the transgenic host cell line, we are able to express more complete recombinant non-secretory N-linked glycoprotein for our customers.

References:

1. Hollister, J.; et al. Engineering the protein n-glycosylation pathway in insect cells for production of biantennary, complex N-glycans. Biochemistry 2002, 41(50): 15093–15104.
2. Chung C., ; et al. Snapshot: N-glycosylation processing pathways across kingdoms. Cell. 2017, 1(171): 258-258.