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Construction, expression and characterization of CD45-immunoglobulin fusion proteins Awrey, Shannon June


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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