A common limitation of the lower eukaryotic expression system is that it can not accurately form N-linked glycans for recombinant glycoprotein as the higher eukaryotic expression system does. This hinders the application of baculovirus systems in the development of therapeutic antibodies.
As experts in biotechnology and protein biology, Creative BioMart and its research team have been committed to solving this problem. Our professional expert team has come up with the solution of Target Protein and Glycosyltransferase Co-expression, which is based on the glycoengineering approach. Through co-infecting the host insect cells with the recombinant viruses harbour target gene and glycozymes sequence, the mammalian-type N-linked glycosylation had been conducted successfully.
In the stage of glycosylation modification, the special β - n - acetylglucosaminidase in wild-type insect cells will trim the non-reducing terminal β 1,2- GlcNAc residue, resulting in the formation of paucimanosidic glycoforms which is different from that of mammalian. Figure 1 shows the difference in N-glycan structure between mammalian and insect cells. Consequentlly, for the practical use, the reform of the biosynthetic pathway of the host insect cell N-glycosylation is required.
Figure 1. The difference in N-glycan structure between mammalian and insect cells.
One of the solution is to co-express the mammalian glycozymes with the target gene. In mammalian cell, non-reducing terminal β 1,2- GlcNAc residue is trimed by β1,4-N-acetylglucosaminyltransferase. Therefore, to mimic the residue processing principle, coding sequence of β1,4-N-acetylglucosaminyltransferase is insert to the vector. The vector harbours the glycosyltransferase gene will be transferred into the host cell to form a stable modified cell line.
The vector harbors β1,4-N-acetylglucosaminyltransferase fragment is transferomed into the insect cell to establish a stable cell line. The vector contains a very early promoter of a baculovirus which can drive the expression of the β1,4-N-acetylglucosaminyltransferase, even in uninfected cells.
Figure 2. β1,4-N-acetylglucosaminyltransferase activity in stably transfected cell lines.The expression of β1,4-N-acetylglucosaminyltransferase is confirmed by an activity assay using a 2-aminopyridine labeled (PA-) agalactosyl biantennary oligosaccharide (GnGn) as an acceptor substrate (Figure 2. A ). However, as shown in Figure 2D, significant activity related to the formation of the bisected oligosaccharide was detected, while no activity was observed in control group cells that had been transfected with the inactive mutant glucosaminyltransferase (Figure 2 B ) or empty vectors (Figure 2 C ).
The result had clearly demonstrated that the mammalian β1,4-N-acetylglucosaminyltransferase is stably expressed in the insect cells and conveys its biology function correctly.
To investigate the effect of β1,4-N-acetylglucosaminyltransferase expression on the oligosaccharide biosynthetic pathway, alterations in N-glycan structures were investigated by a comparison between β1,4-N-acetylglucosaminyltransferase positive Sf21 cells and control (negative) cells. The results showing that N-glycans from β1,4-N-acetylglucosaminyltransferase positive Sf21 cells, but not from parental cells, retain terminal β1,2-GlcNAc residues is consistent with the suggestion that the bisected oligosaccharides are resistant to the cleavage of the non-reducing terminal GlcNAc.