Assuntos
Catepsinas/química , Cisteína Endopeptidases/química , Paragonimus/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Catepsina F , Catepsinas/análise , Catepsinas/genética , Clonagem Molecular , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/genética , Cães , Imuno-Histoquímica , Dados de Sequência Molecular , Paragonimus/genética , Alinhamento de SequênciaRESUMO
Cysteine proteinases play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based approach of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for Clonorchis sinensis, we overexpressed and characterized the 24-kDa cysteine proteinase from adult worms. First, the partial cysteine proteinase gene from C. sinensis was cloned by performing reverse transcription polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA has an open reading frame of 981 bp, and the deduced amino acid sequence shares similarity with the cathepsin L-like cysteine proteinases from Schistosoma mansoni, Paragonimus westermani metacercaria, Fasciola hepatica, and human cathepsin L by 52%, 47%, 34%, and 29%, respectively. The cysteine proteinase was then overexpressed in the yeast Pichia pastoris as an active enzyme on a large-scale basis (19.7 mg/L). The active recombinant enzyme was purified from culture media using a Ni2+-NTA-agarose affinity column and gel filtration chromatography. This 24-kDa recombinant protein exhibited a substrate preference for Z-Phe-Arg-AMC (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amino-4-methyl-coumarin) compared with Z-Arg-Arg-AMC, and the activity was inhibited by E-64 (L-trans-epoxysuccinylleucylamido(4-quanidino)butane).
Assuntos
Catepsinas/biossíntese , Clonorchis sinensis/enzimologia , Cisteína Endopeptidases/biossíntese , Proteínas de Helminto/biossíntese , Proteínas Recombinantes/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina L , Catepsinas/genética , Clonorchis sinensis/genética , Cisteína Endopeptidases/genética , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Helminto/genética , Dados de Sequência Molecular , Pichia/genética , Homologia de Sequência de AminoácidosRESUMO
We constructed a 3'-directed cDNA library of cleistothecia and Hülle cells of Aspergillus nidulans to examine gene expression patterns of the sexual structures and to have probes necessary to isolate sexual structure-specific genes. Sequencing of 360 randomly selected cDNA clones yielded 272 expressed sequence tags (ESTs), most of which probably represent frequently or less expressed genes in sexual structures of A. nidulans. Among the 272 ESTs, 33 ESTs (87 cDNA clones) appeared more than once and 2 ESTs appeared 6 times; 9 ESTs matched GenBank entries. When compared with sequences obtained from a mycelial 3'-directed cDNA library of A. nidulans, 28 out of 33 ESTs seem to be sexual structure-specific. Northern blot analyses of 20 ESTs showed that 17 are sexual structure-specific. The remaining three ESTs also hybridized with RNA isolated from vegetative mycelia. These results suggest that analyses of ESTs from different cell types or tissues can readily demonstrate gene expression patterns of specific cell types and identify cell type-specific cDNA probes.
