Expression and spectroscopic analysis of a mutant hepatitis B virus onco-protein HBx without cysteine residues

Autor(es)

Rui, Edmilson ; Moura, Patricia Ribeiro de ^[2] ; Gonçalves, Kaliandra de Almeida ; Kobarg, Jörg

• Tipo de documento: Artigo Científico
• Fonte: Journal of Virological Methods; v. 126, n. 1/2, p. 65-74, June 2005.
• Área do conhecimento: Ciências Biológicas - Bioquímica
• Assunto(s): Hepatite B Genomas

Resumo

Chronic infection of the hepatitis B virus (HBV) is one of the causes leading to liver cancer. The 3.2 kb genome of HBV encodes four proteins: core antigen, surface antigen, a DNA polymerase and the X protein (HBx). The biological functions of HBx are not fully understood. It has been shown that HBx is a potent trans-activator, which activates transcription of many cellular and viral promoters indirectly via protein-protein interactions. These transactivating activities of HBx may contribute to the development of hepatocellular carcinoma. In this paper a truncated mini-HBx(-Cys) (18-142) protein, where the cysteines had been either deleted or substituted by serines, was constructed by site-directed mutagenesis and overexpressed as a 6xHis fusion protein in Escherichia coli. The 6xHis-mini-HBx(-Cys) protein was isolated from inclusion bodies, purified by Ni-affinity chromatography under denaturing conditions and refolded by sequential dialysis. The structure of the 6xHis-mini-HBx(-Cys) protein was analyzed by circular dichroism, fluorescence and one-dimensional NMR spectroscopic assays. The data presented here suggest that HBx is unstructured but has a propensity to gain secondary structure under specific experimental conditions. Its conformational flexibility might partially explain its functional complexity, namely its capacity to interact with a wide array of signaling proteins, transcriptional regulators and nucleic acids. (AU)

• Processo FAPESP: 98/05891-9 - Expressão, purificação e cristalização da proteína HBx do vírus da Hepatite B para sua análise estrutural por difração de raios X
• Beneficiário: Jörg Kobarg
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 03/12272-3 - Expressão da oncoproteína HBx do vírus de Hepatite B para identificação de RNAs ligantes utilizando o método de selex
• Beneficiário: Kaliandra de Almeida Gonçalves
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 01/00554-9 - Análise funcional e estrutural da proteína HBx do vírus da Hepatite B
• Beneficiário: Patricia Ribeiro de Moura
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 01/06552-8 - Mutagênese da proteína HBx do vírus da Hepatite B e caracterização da sua interação com RNA
• Beneficiário: Edmilson Rui
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto