RESUMO
A gene coding for human stefin B was synthesized by the solid-phase phosphite method and cloned in the pUC8 cloning vector. The insert with the verified DNA sequence was subcloned into two expression vectors and expressed in E. coli as a fusion protein with beta-galactosidase and as a native protein. The CNBr cleaved fusion protein and the native recombinant stefin B were inhibitory to papain and reacted with antibodies against human stefin B.
Assuntos
Clonagem Molecular , Cistatinas , Escherichia coli/genética , Genes Sintéticos , Genes , Inibidores de Proteases/genética , Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Cistatina B , Humanos , Dados de Sequência Molecular , Proteínas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Transcrição GênicaRESUMO
A DNA containing the coding sequence for the human cysteine proteinase inhibitor protein cystatin C has been obtained by enzymatic ligation of chemically synthesized deoxyoligonucleotides, using the Khorana ligation method. The 375 bp synthetic gene carries signals for the translation initiation and termination and was expressed in E. coli as a beta-galactosidase fusion protein as well as a secreted protein under the control of the E. coli alkaline phosphatase signal sequence. The secreted hybrid protein was shown to have similar biological properties as the authentic protein isolated from human plasma.
Assuntos
Cistatinas , Escherichia coli/metabolismo , Genes Sintéticos , Proteínas/genética , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Cistatina C , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Biossíntese de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
The cathepsins B, H and L of human origin were isolated in pure form in sufficient quantities for structural characterization. The complete amino acid sequence of human liver cathepsin B was determined. Partial amino acid sequences of the human kidney cathepsin H and L show the highly conserved region around the active site cysteine. The cysteine proteinase inhibitors stefin A, human stefin B and human cystatin C were isolated, characterized and sequenced. Their amino acid sequences are compared with sequences of other protein inhibitors of the stefin and cystatin family, showing a high degree of homology throughout both families. The stefin and cystatin family, together with newly discovered kininogen family belong to the same superfamily of cystatins. The constructed dendrogram shows that the most closely related inhibitors so far sequenced are human stefin B and rat liver TPI.
