RESUMO
Spleen tyrosine kinase (Syk) is an important non-receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high-throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI-2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze-thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr(525)/Tyr(526) in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high-throughput system.
Assuntos
Baculoviridae/genética , Clonagem Molecular/métodos , Dictyostelium/enzimologia , Dictyostelium/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Ensaios de Triagem em Larga Escala/métodos , Proteínas Tirosina Quinases/biossíntese , Proteínas Recombinantes/biossíntese , Dicroísmo Circular , Dictyostelium/virologia , Estabilidade Enzimática , Escherichia coli/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia de Fluorescência , Modelos Moleculares , Fosforilação , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Quinase SykRESUMO
Animal models, primary cell culture systems and permanent cell lines have provided important information on virulence properties of pathogenic microorganisms. Recently, it has been shown that some inherent limitations of such models can be circumvented by using non-vertebrate hosts such as Caenorhabditis elegans, Drosophila melanogaster and Dictyostelium discoideum. These new models are helpful to follow infection processes at the molecular level. Persuasive support comes from the fact that processes such as phagocytosis, cell signaling or innate immunity can be studied in these surrogate hosts. This review describes the establishment and application of each of the three aforementioned and genetically tractable hosts. In addition, we will report on a number of approaches that led to the identification of host cell factors which influence the susceptibility of the hosts to infection.
