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 R. Emmett; Alan G. Marshall; Timothy M. Logan

doi:10.1016/j.pep.2003.10.011

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 R. Emmett, Alan G. Marshall, Timothy M. Logan

Research output: Contribution to journal › Article › peer-review

4 Scopus citations

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 language	English (US)
Pages (from-to)	274-287
Number of pages	14
Journal	Protein Expression and Purification
Volume	33
Issue number	2
DOIs	https://doi.org/10.1016/j.pep.2003.10.011
State	Published - Feb 2004
Externally published	Yes

Keywords

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

ASJC Scopus subject areas

Biotechnology

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10.1016/j.pep.2003.10.011

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@article{52d22a47f32248b1b5b4956955070fd6,

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 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.",

keywords = "FT-ICR, Fourier transform ion cyclotron resonance, Glycoform homogeneity, MALDI, Mass spectrometry, Recombinant glycoprotein, Thy-1",

author = "Promod Mehndiratta and Walton, {Wendy J.} and Hare, {Joan T.} and Silvia Pulido and Gopalakrishnan Parthasarathy and Emmett, {Mark R.} and Marshall, {Alan G.} and Logan, {Timothy M.}",

note = "Funding Information: We thank Rani Dhanarajan, Mark Tway, Margaret Seavy, and Ewa Bienkiewicz for their contributions to this research. The work was supported by NIH 58143 (T.M.L.) and NSF-CHE-99-09502 (A.G.M.).",

year = "2004",

month = feb,

doi = "10.1016/j.pep.2003.10.011",

language = "English (US)",

volume = "33",

pages = "274--287",

journal = "Protein Expression and Purification",

issn = "1046-5928",

publisher = "Academic Press Inc.",

number = "2",

}

TY - JOUR

T1 - Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells

AU - Mehndiratta, Promod

AU - Walton, Wendy J.

AU - Hare, Joan T.

AU - Pulido, Silvia

AU - Parthasarathy, Gopalakrishnan

AU - Emmett, Mark R.

AU - Marshall, Alan G.

AU - Logan, Timothy M.

N1 - Funding Information: We thank Rani Dhanarajan, Mark Tway, Margaret Seavy, and Ewa Bienkiewicz for their contributions to this research. The work was supported by NIH 58143 (T.M.L.) and NSF-CHE-99-09502 (A.G.M.).

PY - 2004/2

Y1 - 2004/2

N2 - 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.

AB - 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.

KW - FT-ICR

KW - Fourier transform ion cyclotron resonance

KW - Glycoform homogeneity

KW - MALDI

KW - Mass spectrometry

KW - Recombinant glycoprotein

KW - Thy-1

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UR - http://www.scopus.com/inward/citedby.url?scp=0842267067&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2003.10.011

DO - 10.1016/j.pep.2003.10.011

M3 - Article

C2 - 14711516

AN - SCOPUS:0842267067

SN - 1046-5928

VL - 33

SP - 274

EP - 287

JO - Protein Expression and Purification

JF - Protein Expression and Purification

IS - 2

ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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