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UBC Theses and Dissertations
Construction, expression and characterization of CD45-immunoglobulin fusion proteins Awrey, Shannon June
Abstract
The aim of this work was to create, express, and characterize fusion proteins consisting of different alternatively spliced exons of murine CD45, a protein tyrosine phosphatase, linked to the heavy chain constant regions of murine immunoglobulin G. CD45-immunoglobulin fusion proteins were secreted as dimers in a relatively pure form using serum free media at an approximate yield of 1.5-4.5 ug / m l , depending on the isoform of CD45 and the cell line in which it was expressed. Fusion proteins secreted by Cos 7 cells had a higher apparent molecular weight by approximately 5-10 kDa than those expressed by X63-Ag8.653 or T28 cells. The interaction of CD45 with putative ligands may be mediated by specific carbohydrate residues on CD45, therefore, the carbohydrate residues expressed on CD45-immunoglobulin fusion proteins were characterized. O-glycosidase digestion and lectin analysis revealed that all fusion proteins were extensively O-glycosylated in a cell-specific manner. Neuraminidase digestion and analysis of subsequent Peanut agglutinin reactivity suggested that fusion proteins secreted by Cos 7 cells expressed more sialic acid when compared to that secreted by X63-Ag8.653 or T28 cells. Thrombin cleavage and PNGase F digestion revealed that the immunoglobulin portion was 34 kDa and the only site of N-linked carbohydrate addition. A l l fusion proteins reacted with anti-CD45 exon-specific antibodies as predicted with the exception of RA3 6B2, a B220-specific antibody that reacted with CD45RABCIg expressed by Cos 7 cells but not with that expressed by X63-Ag8.653 or T28 cells. RA3 6B2 reacted with fusion proteins containing exons A , B, and C inclusive in addition to fusion proteins containing only exon A. RA3 6B2 binding was not affected by neuraminidase treatment, but did correlate to the binding of wheat germ agglutinin. Once expressed and purified, CD45-immunoglobulin fusion proteins can be used as diagnostic tools in immunoadherence and adhesion assays in an attempt to further our understanding of T lymphocyte signalling via the identification an isoform-specific ligand(s) for murine CD45.
Item Metadata
Title |
Construction, expression and characterization of CD45-immunoglobulin fusion proteins
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1996
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Description |
The aim of this work was to create, express, and characterize fusion proteins
consisting of different alternatively spliced exons of murine CD45, a protein tyrosine
phosphatase, linked to the heavy chain constant regions of murine immunoglobulin G.
CD45-immunoglobulin fusion proteins were secreted as dimers in a relatively pure
form using serum free media at an approximate yield of 1.5-4.5 ug / m l , depending on
the isoform of CD45 and the cell line in which it was expressed. Fusion proteins
secreted by Cos 7 cells had a higher apparent molecular weight by approximately 5-10
kDa than those expressed by X63-Ag8.653 or T28 cells.
The interaction of CD45 with putative ligands may be mediated by specific
carbohydrate residues on CD45, therefore, the carbohydrate residues expressed on
CD45-immunoglobulin fusion proteins were characterized. O-glycosidase digestion
and lectin analysis revealed that all fusion proteins were extensively O-glycosylated in a
cell-specific manner. Neuraminidase digestion and analysis of subsequent Peanut
agglutinin reactivity suggested that fusion proteins secreted by Cos 7 cells expressed
more sialic acid when compared to that secreted by X63-Ag8.653 or T28 cells. Thrombin
cleavage and PNGase F digestion revealed that the immunoglobulin portion was 34
kDa and the only site of N-linked carbohydrate addition.
A l l fusion proteins reacted with anti-CD45 exon-specific antibodies as predicted
with the exception of RA3 6B2, a B220-specific antibody that reacted with CD45RABCIg
expressed by Cos 7 cells but not with that expressed by X63-Ag8.653 or T28 cells.
RA3 6B2 reacted with fusion proteins containing exons A , B, and C inclusive in addition
to fusion proteins containing only exon A. RA3 6B2 binding was not affected by
neuraminidase treatment, but did correlate to the binding of wheat germ agglutinin.
Once expressed and purified, CD45-immunoglobulin fusion proteins can be used
as diagnostic tools in immunoadherence and adhesion assays in an attempt to further
our understanding of T lymphocyte signalling via the identification an isoform-specific
ligand(s) for murine CD45.
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Extent |
8260376 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-02-06
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0086985
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1996-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.