Expression of Glycosylated Mucin-like Domain Using Baculovirus Expression System in Silkworm, Bombyx mori

Baculovirus expression systems (BES) are valuable and popular tools for the production of recombinant proteins. Because insect cells have a posttranslational modifications similar to those of mammalian cells, such as glycosylation, phosphorylation, and protein folding, BES are used to produce proteins from higher eukaryotes, for example, canine interferons–a, human serine–threonine kinase 11, and human acidic and basic fibroblast growth factors (Liu et al., 2005; Martinez et al., 2005; Gouveia et al., 2007). As regards the glycosylation in insect cells, the N–glycosylation pathway has been studied with a view to production of human–like N–glycosylated proteins, but there are few reports of O–glycosylation. In this study, to investigate whether the mucin–type O–glycosylation occurs in silkworm, Bombyx mori, we performed recombinant protein expression of a mucin– like protein as a target using BES in silkworm. Mucins are heavily O–glycosylated glycoproteins found in secreted mucous and as transmembrane glycoproteins of the cell surface to serve as a last protective barrier against extracellular environmental factors like low pH or hydrolytic enzymes (Strouss et al., 1992). Mucins have serine and threonine rich domains called variable number of tandem repeat (VNTR) regions. Mucins may have hundreds of O–GalNAc glycans attached to serine or threonine residues in the VNTR regions and these abundant O–glycan chains may comprise 80% of the molecule by weight (Lan et al., 1987). Four main O–glycan core structures are well known in mammals (Daniel., 2009). The most common O–GalNAc glycan is Gal β 1–3GalNAc α–Ser/Thr (Fig. 1). It is termed a Core 1 or T–antigen and forms many longer, more complex structures. Another common structure is Core 2, contains a branching N–acetylglucosamine attached to core 1 (Fig. 1). Core1 and Core 2 O–GalNAc glycans are found in both glycoproteins and mucins from a variety of cells and tissues. Core 3 and Core 4 stuructures (Fig. 1) are found in a few tissues, such as colon (Podolsky., 1985), bronchi (Lamblin., 1984) and salivary glands (Wieruszesk et al., 1987). In insects, Core 1 type mucin has been observed (E Tian et al., 2008), but other type mucins are yet to be detected. Expression of Glycosylated Mucin–like Domain Using Baculovirus Expression System in Silkworm, Bombyx mori