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
Phosphodiesterase plays an important role in regulating inflammatory pathways and T cell function. The development of phosphodiesterase 7 inhibitor may give better efficacy profile over phosphodiesterase 4 inhibitors. However, the recombinant phosphodiesterase 7 is required in large quantity for high-throughput screening of new drugs by in vitro enzymatic assays. In the present study, recombinant human PDE7A1 was expressed in Dictyostelium discoideum under the control of constitutively active actin-15 promoter. The cytosolic localization of the expressed protein was confirmed by immunofluorescence studies. Upto 2 mg of recombinant protein was purified using His-Tag affinity column chromatography followed by ion-exchange Resource Q column purification. The recombinant protein expressed in D. discoideum followed Michaelis-Menten kinetics similar to the protein expressed in mammalian system and showed no major changes in affinity to substrate or inhibitors. Thus, our study clearly demonstrates a robust expression system for successful bulk production of pharmacologically active isoform of human PDE7A1 required for high-throughput assays.