METODO DE EXPRESION DE GENES HETEROLOGOS EN LA LEVADURA PICHIA PASTORIS, VECTORES DE EXPRESION Y MICROORGANISMOS TRANSFORMADOS

• Main Authors: MORALES GRILLO, JUAN, HERRERA MARTINEZ, LUIS SATURNINO, YONG GONZALEZ, VLADIMIR, TORRENS MADRAZO, ISIS DEL CARMEN, DELGADO BOADA, JULIO MARCOS, MARGOLLEZ CLARK, EMILIO
• Format: Patent
• Language: Spanish
• Published: 16.02.2003
• Edition: 7
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; ENZYMOLOGY; FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; ORGANIC CHEMISTRY; PEPTIDES; PROCESSES USING MICROORGANISMS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

LA PRESENTE INVENCION PROPORCIONA UN METODO ALTAMENTE EFICAZ PARA LA EXPRESION DE GENES HETEROLOGOS, USANDO COMO HUESPED UN AUXOTROFICO MUTANTE HIS-3 DE LA CEPA BKM-90 DE "PICHIA PASTORIS" Y COMO ADN RECOMBINANTE PARA LA TRANSFORMACION DEL MISMO, UN VECTOR INTEGRADOR QUE CONTIENE UN MARCADOR DE SELECCION FUNCIONAL DE "SACAROMICES CEREVISIAE", PROMOTOR DEL GEN ALCOHOL OXIDASA (AOX SUB 1) DE LA PROPIA LEVADURA, COMO DETERMINADOR DE LA TRANSCRIPCION EL DE LA GLICERALDEHIDO 3-FOSFATO DESHIDROGENASA (GAP) DE "S. CEREVISIAE", O EL QUE LLEVA EL GEN HETEROLOGO QUE VAMOS A EXPRESAR, Y OTRA SECUENCIA HOMOLOGA AL GENOMA DE "PICHIA PASTORIS", QUE SIRVE PARA LA INTEGRACION DEL MISMO. LOS VECTORES DE EXPRESION USADOS POSTERIORMENTE CONTIENEN UN GEN HETEROLOGO UNIDO AL PEPTIDO INDICADO DEL GEN DE LA "SUCROSA INVERTASA" (SUC2) DE "S. CEREVISIAE". ESTE METODO GARANTIZA QUE LOS PRODUCTOS DE POLIPEPTIDOS SON RECUPERABLES EN CONDICIONES CONTROLABLES Y CON ALTOS RENDIMIENTOS. LAS CEPAS DE LA PRESENTE INVENCIONPUEDEN SER UTILIZADAS PARA LA PRODUCCION A GRAN ESCALA DE PRODUCTOS FARMACEUTICOS O DE ENZIMAS DE INTERES INDUSTRIAL. The present invention provides a highly efficient method for the expression of heterologous genes, using as the host an auxotrophic his3 mutant of strain BKM-90 of Pichia pastoris and as recombinant DNA for the transformation thereof an integrative vector which contains a functional selection marker of Saccharomyces cerevisiae, the promotor of the alcohol oxidase gene (AOX1) of the yeast itself, as the transcription terminator that of glyceraldehyde 3-phosphate dehydrogenase (GAP) of S. cerevisiae, or that which is carried by the heterologous gene to be expressed, and another sequence homologous to the genome of Pichia pastoris which serves for the integration thereof. The expression vectors used further contain a heterologous gene bound to the signal peptide of the sucrose invertase gene (SUC2) of S. cerevisiae. This method guarantees that the polypeptide products are recoverable under controllable conditions and with high yields. The strains according to the present invention can be used for the large-scale production of pharmaceutical products or of enzymes of industrial interest.