RESUMO
Agkistin was a new snake venom metalloproteinase (SVMP) gene which was cloned from Agkistrodon halys. Its deduced amino acid sequence has two additional cysteines (Cys407 and Cys426) in the disintegrin domain compared to other RGD containing SVMPs. The full-length gene (Agkistin) and its disintegrin region (named Agkistin-s) were expressed by baculovirus expression system (pFastBac-Htb vector) with His-tag, and their platelet aggregation-inhibition activity was evaluated. The expressed protein Agkistin can also induce apoptosis of HMEC cells in the basal medium after incubated at 37 degrees C for 20 h.
Assuntos
Agkistrodon/metabolismo , Venenos de Crotalídeos/metabolismo , Metaloendopeptidases/metabolismo , Venenos de Serpentes/enzimologia , Agkistrodon/genética , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Sequência de Bases , Clonagem Molecular , Venenos de Crotalídeos/química , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/isolamento & purificação , Citometria de Fluxo , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/isolamento & purificação , Dados de Sequência Molecular , Agregação Plaquetária/fisiologia , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The cDNA encoding the mature annexin V was isolated by using RT-PCR method from total RNAs of fresh human placenta. The result of sequencing indicated that the sequence of isolated annexin V cDNA was the same as the reported nucleotide sequence of annexin V. The annexin V cDNA was cloned into expression plasmid pET24a(+) under T7 promoter and then transformed into E.coli BL21(DE3). SDS-PAGE analysis revealed that the human annexin V was highly expressed and accumulated up to 38% of total bacterial proteins in soluble form after the induction by 1 mmol/L IPTG. The purified human annexin V can significantly prolong activated partial thromboplastin time (APTT).
