Title : Expression, purification, and characterization of avian
Thy-1 from
Lec1 mammalian and Tn5 insect cells
Abstract :
- Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites
- Herein, we report the cloning of a novel isoform of avian Thy-1 and the subsequent expression, purification, and characterization of a soluble form of Thy-1 from Lec1 mammalian and Tn5 insect cells
- The novel isoform of Thy-1 isoform of Thy-1 differs from the previously reported chicken isoform by eight amino acid residues , but these changes do not alter the secondary structure content, the disulfide bond pattern, or the sites of glycosylation
- The disulfide linkage pattern and glycoform distribution on each N-glycosylation site of recombinant chicken Thy-1 from both cell lines were determined by a combination of amino-terminal sequencing and mass spectrometry
- The mass spectral data showed that the amino-terminal glutamine was modified to pyroglutamate
- Recombinant Thy-1 from Lec1 cells contained (GlcNAc)(2)(Man)(5) on asparagine 60 , whereas the oligosaccharides on asparagine 23 and 100 contained approximately 80% (GlcNAc)(2)(Man)(4) and approximately 20% (GlcNAc)(2)(Man)(5)
- The glycoforms on Thy-1 expressed in Tn5 cells were more heterogeneous, with the oligosaccharides ranging over (GlcNAc)(2)(Fuc)(0-2)(Man)(2-3) on each site
- The ability to generate recombinant glycoproteins with restricted carbohydrate heterogeneity is the first step toward the systematic study of structure-function relationships in intact glycoproteins