High level expression and characterization of a truncated soluble murine Fc gamma receptor.
Item
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Title
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High level expression and characterization of a truncated soluble murine Fc gamma receptor.
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Identifier
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AAI9119670
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identifier
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9119670
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Creator
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Qu, Zhengxing.
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Contributor
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Adviser: Jay C. Unkeless
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Date
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1991
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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An expression vector encoding a truncated secreted moFc{dollar}\gamma{dollar}RII{dollar}\beta{dollar}(tFc{dollar}\gamma{dollar}RII{dollar}\beta{dollar}) protein was constructed by deletion of transmembrane and cytoplasmic domains from an Fc{dollar}\gamma{dollar}RII{dollar}\beta{dollar}2 cDNA clone, and insertion of the truncated cDNA into the eukaryotic expression vector, pcEXV-3. The DNA construct was transfected into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and the production of Fc{dollar}\gamma{dollar}RII{dollar}\beta{dollar} was then amplified by gradual addition of methotrexate. The resulting cell line, D1959, secretes 2-3 {dollar}\mu{dollar}g/ml/24 h of truncated Fc{dollar}\gamma{dollar}RII{dollar}\beta{dollar}, which has a M{dollar}\sb{lcub}\rm r{rcub}{dollar} of 30,000-35,000 on SDS-PAGE and reacts with anti-Fc{dollar}\gamma{dollar}R antibodies on immunoblots. The truncated Fc{dollar}\gamma{dollar}RII{dollar}\beta{dollar} was purified to homogeneity by affinity chromatography on IgG-Sepharose.;The truncated Fc{dollar}\gamma{dollar}RII purified from the medium conditioned by D1959 cells was further characterized. The truncated Fc{dollar}\gamma{dollar}RII{dollar}\beta{dollar} is glycosylated and sialylated and shows a high degree of size and charge heterogeneity on SDS-PAGE and IEF. Titration with Ellman's reagent revealed that the 4 cysteine residues in the protein form 2 disulfide bonds. As dose intact Fc{dollar}\gamma{dollar}RII, the truncated Fc{dollar}\gamma{dollar}RII was shown to be capable of binding IgG1, IgG2a and IgG2b immune complexes. The binding of the immune complexes was somewhat better at more acidic pH. The glycosylation of tFc{dollar}\gamma{dollar}RII is not required for the binding activity since both aglycosylated and deglycosylated tFc{dollar}\gamma{dollar}RII bind with IgG-coated beads.;Crosslinking of the truncated Fc{dollar}\gamma{dollar}RII with a heterobifunctional crosslinker, SASD, provided evidence that, although tFc{dollar}\gamma{dollar}RII exists as monomer in solution, there is interaction between tFc{dollar}\gamma{dollar}RII molecules. Upon irradiation to activate the azido moiety, SASD derivatized Fc{dollar}\gamma{dollar}RII monomers form very large complexes. The extent and rate of formation of these complexes is independent on the concentration of tFc{dollar}\gamma{dollar}RII{dollar}\beta{dollar} in the range of 3 {dollar}\mu{dollar}M to 0.3 {dollar}\mu{dollar}M. Complex formation does not occur after denaturation of the protein with SDS.;The truncated Fc{dollar}\gamma{dollar}RII was reactive with mAbs elicited by immunization with intact Fc{dollar}\gamma{dollar}RII, with the exception of 6B7C. Since the tFc{dollar}\gamma{dollar}RII is lacking 8 amino acid residues proximal to the transmembrane domain, we hypothesized that the 6B7C epitope encompassed in the deleted sequence. By binding of mAb 6B7C to a synthetic peptide conjugate, the 6B7C epitope was mapped to residues 169-183 of the intact Fc{dollar}\gamma{dollar}RII{dollar}\beta{dollar}.;With the availability of a large quantity of purified tFc{dollar}\gamma{dollar}RII protein, we have attempted to crystallize the truncated Fc{dollar}\gamma{dollar}RII for X-ray crystallography. The fully glycosylated tFc{dollar}\gamma{dollar}RII crystallized very poorly from an oil. However, the partially deglycosylated protein crystallized readily. Single crystals were grown to approximate 1.0 {dollar}\times{dollar} 0.2 {dollar}\times{dollar} 0.2 mm in size and diffracted to 3 A resolution. Further studies to obtain high quality crystals for deglycosylated and neuraminidase-treated tFc{dollar}\gamma{dollar}RII are underway.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
