RESUMEN
An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co-express both the L (preS1-S2-S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol-inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5-8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle-specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H polymorpha-derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Antígenos de Superficie de la Hepatitis B/biosíntesis , Pichia/genética , Secuencia de Bases , Western Blotting , Medios de Cultivo , ADN de Hongos/química , Fermentación , Vectores Genéticos , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Immunoblotting , Datos de Secuencia Molecular , Pichia/metabolismo , Plásmidos , Regiones Promotoras Genéticas , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Mapeo RestrictivoRESUMEN
A host/vector system suitable for large-scale production of HBsAg has been constructed and optimized in terms of the expression plasmid and yeast host strain in order to permit fermentation to very high cell densities. The final expression plasmid contains the coding sequence of the major HBsAg protein (P24) flanked by the promoter sequences from a glycolytic gene and by the transcription-termination region of the ARG3 gene. The host/vector system was found to be genetically stable under large-scale fermentation conditions as demonstrated by nucleotide sequencing and restriction mapping experiments. The P24 protein is recovered from yeast as particles whose physiochemical properties are very similar to those of plasma-derived HBsAg.
Asunto(s)
Ingeniería Genética/métodos , Vectores Genéticos , Antígenos de Superficie de la Hepatitis B/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Clonación Molecular , ADN Recombinante/inmunología , Plásmidos , Transcripción GenéticaRESUMEN
The production in yeast cells of the recombinant DNA hepatitis B vaccine of SmithKline Biologicals involves an optimized fermentation process followed by cell disruption and extraction, together with other soluble yeast components of the surface antigen of the hepatitis B virus. The subsequent purification process includes precipitation steps, ion exchange and gel permeation chromatography, and caesium chloride ultracentrifugation. The yeast-derived antigen occurs as spherical particles containing the non-glycosylated HBsAg polypeptide, lipid, and Tween 20. The purity of the polypeptide is above 95% and confirmed by the absence of an immune response to yeast-derived contaminants in vaccinees. Yeast DNA levels were less than 10 pg/vaccine dose. Various biochemical analyses showed that the recombinant polypeptide was faithfully expressed and did not undergo unwanted processing or degradation during fermentation or purification. These results indicate that the recombinant HBsAg can be effectively produced in yeast and processed to a high degree of purity to yield HBsAg particles displaying most of the characteristic properties of plasma-derived HBsAg.
Asunto(s)
Antígenos , Antígenos de Superficie de la Hepatitis B , Hepatitis B/prevención & control , Saccharomyces cerevisiae/genética , Vacunas Sintéticas , Secuencia de Aminoácidos , Anticuerpos Antiidiotipos/análisis , Antígenos/inmunología , ADN de Hongos/análisis , ADN Recombinante/inmunología , Antígenos de Superficie de la Hepatitis B/genética , Humanos , Inmunoglobulina E/inmunología , Lípidos/análisis , Microscopía Electrónica , Proteínas/análisis , Vacunación , Vacunas Sintéticas/inmunologíaRESUMEN
The cDNA coding for mature human alpha 1-proteinase inhibitor (alpha 1-PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated alpha 1-PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2mu and the leu2D selection marker. Biologically active recombinant alpha 1-PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high-Mr yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE-agarose, Zn-chelate agarose, kappa-chain agarose, heparin-agarose and aminohexyl-agarose.
Asunto(s)
Proteínas Sanguíneas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica , Humanos , Plásmidos , Levaduras , alfa 1-AntitripsinaRESUMEN
Yeast synthesizes the surface antigen protein of Hepatitis B virus when the structural gene is fused to the promoter from the ARG3 gene. Analysis of extracts and total cells shows that the primary translation product can be identified as a poorly antigenic monomer with an estimated molecular weight of 22K. In cell extracts Y-HBsAg is in the form of 20 nm particles which, like serum derived particles, are highly immunogenic in mice and monkeys. Yeast derived surface antigen is thus a viable alternative to the present serum derived HBV vaccines.
Asunto(s)
ADN Recombinante , Antígenos de Superficie de la Hepatitis B/genética , Levaduras/metabolismo , Animales , Centrifugación por Gradiente de Densidad , Chlorocebus aethiops , Electroforesis en Gel de Poliacrilamida , Antígenos de Superficie de la Hepatitis B/análisis , Antígenos de Superficie de la Hepatitis B/inmunología , RadioinmunoensayoRESUMEN
Nucleotide sequence and deletion analysis have been used to identify the regulatory and coding sequences comprising the cholera toxin operon (ctx). Incorporation of defined in vitro-generated ctx deletion mutations into Vibrio cholerae by in vivo genetic recombination produced strains which have practical value in cholera vaccine development.
Asunto(s)
Toxina del Cólera/genética , Vacunas contra el Cólera , Recombinación Genética , Secuencia de Bases , Clonación Molecular , Regulación de la Expresión Génica , Operón , Vibrio cholerae/genéticaRESUMEN
The genomes of HBV viruses of two different serotypes were cloned in E. coli. Sequences coding for the major polypeptides of surface antigen (HBsAg) were fused with the 5' end of a cloned yeast arg3 gene. When introduced into yeast, on a suitable vector, the hybrid gene directed the synthesis of a fusion protein. Crude extracts of such strains were shown to contain HBsAg like material having physical properties characteristic of the antigen isolated from the plasma of chronic human carriers, as judged by isopycnic and rate zonal centrifugation. Furthermore, these extracts readily elicit specific anti-HBsAg antibodies in rabbits. Further manipulations of the 5' part of the arg3 gene resulted in the introduction of a unique restriction site located in the 5' non translated leader sequence. The resulting vector was used to construct a recombinant plasmid directing the synthesis of the mature (226 amino acids) HBsAg polypeptide.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/genética , Levaduras/metabolismo , Antígenos de Superficie de la Hepatitis B/inmunología , Plásmidos , Recombinación GenéticaAsunto(s)
Toxinas Bacterianas/genética , Enterotoxinas/genética , Escherichia coli/genética , Genes , Bioensayo , Sistema Libre de Células , Clonación Molecular , Elementos Transponibles de ADN , ADN Bacteriano/genética , ADN Recombinante , Peso Molecular , Plásmidos , Precursores de Proteínas/genéticaRESUMEN
Density transfer analysis of deoxyribonucleic acid from Bacillus subtilis 168 thy spores germinating in 5-bromouracil medium shows the order of replication of genetic markers to be: purA16, cysA14, sacA, ctrA, (narB, arol), dal, (hisA1, purB6), (tre-12, thr-5), (argA, aroG, argC4), (metC, leu-8, pheA), (ura-1, aroD), lys-1, (trpC, metB, ilvA, citB, citK, gltA). The precise order of transfer of markers within parentheses could not be determined in these experiments. Taken together with new PBS1 transduction data presented here and in the accompanying paper of J. Lepesant-Kejzlarová, J.-A. Lepesant, J. Walle, A. Billaut, and R. Dedonder (1975), the results can be resolved in terms of a symmetric, fully bidirectional mode of chromosome replication with a replication origin close to the purA16 marker and a terminus in the region of the gltA, citK loci, diametrically opposed to the origin. A new genetic map of the B. subtilis 168 chromosome is presented.