公开(公告)号：WO1990003431A1

公开(公告)日：1990-04-05

申请号：PCT/US1989004164

申请日：1989-09-25

Applicant: THE SALK INSTITUTE BIOTECHNOLOGY/INDUSTRIAL ...

Inventor： THE SALK INSTITUTE BIOTECHNOLOGY/INDUSTRIAL ... ,  BRIERLEY, Russell, Arthur ,  SIEGEL, Robert, Steven ,  BUSSINEAU, Christopher, Michael ,  CRAIG, William, Scot ,  HOLTZ, Gregory, Clyde ,  DAVIS, Geneva, Ruth ,  BUCKHOLZ, Richard, Gordon ,  THILL, Gregory, Patrick ,  WONDRACK, Lillian, Margaret ,  DIGAN, Mary, Ellen ,  HARPOLD, Michael, Miller ,  LAIR, Stephen, Vernon ,  ELLIS, Steven, Bradley ,  WILLIAMS, Mark, Edward

IPC: C12N05/00

CPC classification number: C12N9/0089 ,  C07K14/485 ,  C12N9/2462 ,  C12N15/815

Abstract: The invention provides a method of increasing the production of a recombinant gene product from a culture of a recombinant methylotrophic yeast host, wherein said product is made by expression from a recombinant gene sequence operably associated with a methanol-responsive expression control element. In the method of the invention, the methylotrophic yeast host is first cultured on a medium with a high concentration of multi-carbon, carbon-source nutriment, such as glycerol, but with little or no methanol, in order to increase the density of the host cells with little or no expression of the recombinant gene product. When the host cells have achieved a suitable density in the culture medium, the culture is subjected to a phase during which the concentration of multi-carbon, carbon-source nutriments is maintained sufficiently low that the methanol-responsive control element controlling expression of the recombinant gene encoding the desired product is derepressed. Finally, the culture is subjected to a phase of high production of the recombinant gene product by increasing the concentration of methanol while maintaining the concentration of multi-carbon, carbon-source nutriments at a low level. The invention is illustrated with production, using Pichia pastoris, of bovine lysozyme c2, human lysozyme, human epidermal growth factors (1-52) and (1-48), and human superoxide dismutase.

Abstract translation: 本发明提供了从重组甲基营养酵母宿主的培养物中增加重组基因产物的产生的方法，其中所述产物通过与甲醇应答表达控制元件可操作地相关的重组基因序列的表达来制备。 在本发明的方法中，甲基营养酵母宿主首先在具有高浓度多碳，碳源营养物质的甘油，但几乎不用甲醇或不含甲醇的培养基上培养，以增加其浓度 具有很少或没有表达重组基因产物的宿主细胞。 当宿主细胞在培养基中达到合适的密度时，培养物经历一个阶段，在此阶段，多碳，碳源营养物质的浓度保持足够低，以致控制重组体的表达的甲醇应答控制元件 编码所需产物的基因是去除的。 最后，通过提高甲醇的浓度，同时保持多碳，碳源营养浓度低的水平，使培养物进入高产量的重组基因产物。 使用巴斯德毕赤酵母，牛溶菌酶c2，人溶菌酶，人表皮生长因子（1-52）和（1-48）和人超氧化物歧化酶的生产说明本发明。

公开(公告)号：WO1989004320A1

公开(公告)日：1989-05-18

申请号：PCT/US1988003907

申请日：1988-11-02

Applicant: THE SALK INSTITUTE BIOTECHNOLOGY/INDUSTRIAL ...

Inventor： THE SALK INSTITUTE BIOTECHNOLOGY/INDUSTRIAL ... ,  DIGAN, Mary, Ellen ,  HARPOLD, Michael, Miller ,  LAIR, Stephen, Vernon ,  THILL, Gregory, Patrick ,  SIEGEL, Robert, Steven ,  ELLIS, Steven, Bradley ,  WILLIAMS, Mark, Edward

IPC: C07H15/12

CPC classification number: C12N9/2462 ,  C07K2319/02 ,  C12N15/815

Abstract: The present invention provides a method of producing at high levels an animal lysozyme c by culturing Pichia pastoris cells, which have a gene capable of expressing the pre-form of the animal lysozyme c in such cells, under conditions such that the gene is transcribed. Mature animal lysozyme c is secreted at a high level from such cells into the culture medium, when the cells are cultured under conditions such that the animal pre-lysozyme c gene is transcribed in them. Also provided by the invention are DNAs capable of transforming Pichia pastoris to express animal pre-lysozyme c's, cultures of P. pastoris cells transformed with such DNAs, bovine pre-lysozyme c2, the signal peptide of bovine pre-lysozyme c2, and DNAs encoding those polypeptides.

Abstract translation: 本发明提供一种通过培养巴斯德毕赤酵母细胞，在具有能够在该细胞中表达动物溶菌酶c的形式的基因的基因被转录的条件下高水平生产动物溶菌酶的方法。 当细胞在其中转录动物前溶菌酶c基因的条件下培养细胞时，成熟的动物溶菌酶c从这样的细胞分泌到培养基中。 本发明还提供能够转化巴斯德毕赤酵母以表达动物前溶菌酶c的DNA，用这种DNA转化的巴斯德毕赤酵母细胞的培养物，牛前溶菌酶c2，牛前溶菌酶c2的信号肽和编码 那些多肽。