RESUMO
Human JNK stimulatory phosphatase-1 (JSP-1) is a novel member of dual specificity phosphatases. A C-terminus truncated JSP-1 was expressed in Escherichia coli and was crystallized using the sitting-drop vapor diffusion method. Thin-plate crystals obtained at 278 K belong to a monoclinic space group, C2, with unit-cell parameters a = 84.0 A, b = 49.3 A, c = 47.3 A, and beta = 119.5 degrees , and diffract up to 1.5 A resolution at 100 K. The structure of JSP-1 has a single compact (alpha/beta) domain, which consists of six alpha-helices and five beta-strands, and shows a conserved structural scaffold in regard to both DSPs and PTPs. A cleft formed by a PTP-loop at the active site is very shallow, and is occupied by one sulfonate compound, MES, at the bottom. In the binary complex structure of JSP-1 with MES, the conformations of three important segments in regard to the catalytic mechanism are not similar to those in PTP1B. JSP-1 has no loop corresponding to the Lys120-loop of PTP1B, and tryptophan residue corresponding to the substrate-stacking in PTP1B is substituted by alanine residue in JSP-1.
Assuntos
Fosfoproteínas Fosfatases/química , Proteínas Tirosina Fosfatases/química , Ácidos Alcanossulfônicos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Fosfatases de Especificidade Dupla , Humanos , Camundongos , Fosfatases da Proteína Quinase Ativada por Mitógeno , Modelos Moleculares , Dados de Sequência Molecular , Morfolinas/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/isolamento & purificação , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Proteína Fosfatase 1 , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/isolamento & purificação , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The automation of SDS-PAGE required substantial investment. To lower this barrier, we devised a cost-effective, simple, and general method where samples are loaded on SDS-PAGE gels held by a newly devised "Gel Adaptor" on a general automated liquid handler. In this method, the liquid handler loads samples on the SDS-PAGE gels held at an angle of 80 degrees on the Gel Adaptor so that the samples can be vertically loaded on the narrow paths of the wells of the slanted gels. This method allows the automated liquid handler to load 144 samples on SDS-PAGE gels in approximately 10 min, greatly reducing the time and cost for high-throughput SDS-PAGE analyses.