Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells

Promod Mehndiratta, Wendy J. Walton, Joan T. Hare, Silvia Pulido, Gopalakrishnan Parthasarathy, Mark Emmett, Alan G. Marshall, Timothy M. Logan

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 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-glysosylation 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 ∼80% (GlcNAc)2(Man)4 and ∼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.

Original languageEnglish (US)
Pages (from-to)274-287
Number of pages14
JournalProtein Expression and Purification
Volume33
Issue number2
DOIs
StatePublished - Feb 2004
Externally publishedYes

Fingerprint

Purification
Insects
Asparagine
Oligosaccharides
Glycosylation
Glycoproteins
Protein Isoforms
Disulfides
Chickens
Pyrrolidonecarboxylic Acid
Cloning
Glutamine
Mass spectrometry
Organism Cloning
Mass Spectrometry
Cells
Carbohydrates
Amino Acids
Cell Line
N,N-diacetylchitobiose

Keywords

  • Fourier transform ion cyclotron resonance
  • FT-ICR
  • Glycoform homogeneity
  • MALDI
  • Mass spectrometry
  • Recombinant glycoprotein
  • Thy-1

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells. / Mehndiratta, Promod; Walton, Wendy J.; Hare, Joan T.; Pulido, Silvia; Parthasarathy, Gopalakrishnan; Emmett, Mark; Marshall, Alan G.; Logan, Timothy M.

In: Protein Expression and Purification, Vol. 33, No. 2, 02.2004, p. 274-287.

Research output: Contribution to journalArticle

Mehndiratta, P, Walton, WJ, Hare, JT, Pulido, S, Parthasarathy, G, Emmett, M, Marshall, AG & Logan, TM 2004, 'Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells', Protein Expression and Purification, vol. 33, no. 2, pp. 274-287. https://doi.org/10.1016/j.pep.2003.10.011
Mehndiratta, Promod ; Walton, Wendy J. ; Hare, Joan T. ; Pulido, Silvia ; Parthasarathy, Gopalakrishnan ; Emmett, Mark ; Marshall, Alan G. ; Logan, Timothy M. / Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells. In: Protein Expression and Purification. 2004 ; Vol. 33, No. 2. pp. 274-287.
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