RESUMO
The trpE (or anthranilate synthetase) gene product has been used extensively as a fusion protein for the expression of a myriad of biologically active proteins. A trpE construct can be produced in high yield, is relatively resistant to proteolysis, and separates from the bulk of E. coli proteins because of its insolubility. We have isolated and characterized a monoclonal antibody against the TrpE protein for use as a detection and immunoaffinity reagent. The MAb, TRP 7.4, is highly specific for the TrpE protein and has a relative affinity of 1.0 ng. The antibody can also be used to detect TrpE constructs on Western blots. In addition, TRP 7.4 has been used to purify a TrpE-IL-6 fusion protein. These studies show the utility of this MAb as a tool for both research and protein purification.
Assuntos
Antranilato Sintase/isolamento & purificação , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Interleucina-6/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Antranilato Sintase/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Proteínas de Bactérias/imunologia , Western Blotting , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/imunologia , Técnicas de Imunoadsorção , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Proteínas Recombinantes de Fusão/imunologia , SolubilidadeRESUMO
Autoimmune MRL-lpr/lpr mice develop an SLE-like disease characterized by a profound lymphadenopathy within an L3T4-, Lyt-2- (DN), B220+ T-cell population. Despite its immature phenotype this subset expresses mature alpha beta TCR belonging predominantly to the V beta 8 gene family and appears to be identical to an activated form of a minor T cell population present in both the thymus and periphery of normal mice. However, the mechanisms underlying the greatly increased cellularity in lpr/lpr-bearing mice are not understood. In this study, the IL-2R expression of lpr/lpr T cells was examined to assess the contribution of IL-2-mediated division to their expansion. The lpr/lpr DN T cells lacked high-affinity IL-2R, even after stimulation, suggesting that IL-2-dependent proliferation plays no role in the expansion of these cells and demonstrating the existence of this unusual T cell phenotype in vivo.
