RESUMO
A 1.3-kb segment of Escherichia coli DNA containing the regulatory gene, araC, and the promoter of the araBAD operon was amplified by the polymerase chain reaction (PCR) and cloned into pUC18, resulting in plasmid pKB130 that produced the alpha fragment of beta-galactosidase upon addition of L-arabinose (L-ara). A synthetic gene for human immunodeficiency virus (HIV)-1 preprotease was placed downstream of the ara-BAD promoter in pKB130 to create a translational fusion inducible by addition of L-ara. The fusion protein correctly autoprocessed in vivo to yield a mature 99-amino-acid HIV-1 protease, which was found predominantly in inclusion bodies. This material could be refolded to an active form, which was purified to homogeneity. A small fraction of the protease was expressed in vivo as a soluble active form, which allowed the monitoring of expression during fermentation by a rapid and simple whole cell assay employing an HIV-1 protease-specific fluorogenic substrate.
Assuntos
Escherichia coli/genética , Protease de HIV/biossíntese , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Arabinose/genética , Sequência de Bases , Western Blotting , Escherichia coli/enzimologia , Escherichia coli/ultraestrutura , Protease de HIV/genética , Protease de HIV/isolamento & purificação , Microscopia Eletrônica , Dados de Sequência Molecular , Óperon/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Mapeamento por RestriçãoRESUMO
The gene coding for the major outer capsid protein (VP7) of simian rotavirus SA-11 has been expressed in a baculovirus-insect cell system. The resulting protein is 35 kDa and is primarily associated with the endoplasmic reticulum. Neutralizing SA-11 polyclonal antiserum and VP7 monospecific antiserum reacted specifically with the expressed gene product. Antiserum derived against the recombinant VP7 protein neutralized SA-11 rotavirus infectivity in a fluorescent focus assay.
Assuntos
Baculoviridae/genética , Proteínas do Capsídeo , Capsídeo/genética , Genes Virais , Vetores Genéticos , Rotavirus/genética , Proteínas Estruturais Virais/genética , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Sequência de Bases , Capsídeo/imunologia , Clonagem Molecular , DNA Viral/química , Cobaias , Dados de Sequência Molecular , Mariposas/genética , Testes de Neutralização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Mapeamento por Restrição , Rotavirus/imunologia , Integração ViralRESUMO
A procedure is described which employs pepstatin-agarose for the affinity purification of either HIV-1 or HIV-2 protease from two similar recombinant E. coli constructs that were developed for the expression of these enzymes. HIV-2 protease was routinely expressed at much higher levels than the HIV-1 enzyme and pepstatin-agarose was the only chromatography step required to isolate pure HIV-2 protease from crude bacterial lysates. A Mono S ionic exchange step following pepstatin-agarose chromatography was sufficient to bring the HIV-1 protease to homogeneity. Purification of either enzyme can be completed in several days yielding homogeneous preparations suitable for crystallization and other physical characterization.
