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1. 发明授权

US5827684A Production of Bacillus entomotoxins in methylotrophic yeast 失效
标题翻译：甲基营养酵母中芽孢杆菌毒素的生产
公开(公告)号：US5827684A
公开(公告)日：1998-10-27
申请号：US231342
申请日：1994-04-19
申请人： Kotikanyadanam Sreekrishna , William D. Prevatt , Gregory P. Thill , Geneva R. Davis , Patricia Koutz , Kathryn A. Barr , Sharon A. Hopkins
发明人： Kotikanyadanam Sreekrishna , William D. Prevatt , Gregory P. Thill , Geneva R. Davis , Patricia Koutz , Kathryn A. Barr , Sharon A. Hopkins
IPC分类号： A01N63/00 , A01N63/02 , A01N63/04 , C07K14/32 , C12N1/19 , C12N15/09 , C12N15/81 , C12P21/02 , C12R1/07 , C12R1/84 , C12N15/32 , C12N15/63 , C12N21/02
CPC分类号： C12N15/815 , A01N63/04 , C07K14/32
摘要： A method for producing one or more Bacillus toxin polypeptides by culturing methylotrophic yeast cells which have a gene(s) capable of expressing the Bacillus toxin polypeptide(s) in such cells under conditions that the gene(s) is/are transcribed is provided. The toxin polypeptide encoding segment of the gene(s) has a G+C content of about 40%-55%, and preferably comprises methylotrophic yeast codons. The preferred species of yeast for expressing such synthetic Bacillus toxin gene(s) is Pichia pastoris. Bacillus toxin polypeptides encoded by synthetic genes are expressed at high levels in transformed methylotrophic yeast cells. The toxin expressing cells may be administered as live cells or heat-killed whole cells to provide an insecticidal composition for killing susceptible insect larvae. Also provided by the present invention are DNAs capable of transforming methylotrophic yeast to express one or more Bacillus toxin polypeptides, cultures of such yeast cells transformed with such DNAs and novel Bacillus toxin polypeptides made by the method of the invention. The transformed yeast cells of the present invention are readily ingested as food by insect larvae which are susceptible to the toxin polypeptides.
摘要翻译：提供了一种通过培养具有能够在基因被转录的条件下能够在这些细胞中表达芽孢杆菌毒素多肽的基因的甲基营养酵母细胞产生一种或多种芽孢杆菌毒素多肽的方法。编码基因片段的毒素多肽的G + C含量为约40％-55％，优选包含甲基营养酵母密码子。用于表达这种合成芽孢杆菌毒素基因的酵母的优选种类是巴斯德毕赤酵母。由合成基因编码的芽孢杆菌毒素多肽在转化的甲基营养酵母细胞中以高水平表达。表达毒素的细胞可以作为活细胞或热灭活的全细胞施用，以提供用于杀灭易感昆虫幼虫的杀虫组合物。本发明还提供能够转化甲基营养酵母以表达一种或多种芽孢杆菌毒素多肽的DNA，用这种DNA转化的这种酵母细胞的培养物和通过本发明的方法制备的新的芽孢杆菌毒素多肽。本发明的转化酵母细胞易于被毒素多肽易感的昆虫幼虫摄取为食物。

2. 发明授权

US5612198A Production of insulin-like growth factor-1 in methylotrophic yeast cells 失效
标题翻译：在甲基营养酵母细胞中生产胰岛素样生长因子-1
公开(公告)号：US5612198A
公开(公告)日：1997-03-18
申请号：US308196
申请日：1994-09-19
申请人： Russell A. Brierley , Geneva R. Davis , Gregory C. Holtz , Martin A. Gleeson , Bradley D. Howard
发明人： Russell A. Brierley , Geneva R. Davis , Gregory C. Holtz , Martin A. Gleeson , Bradley D. Howard
IPC分类号： C07K14/65 , C12N15/81 , C12N15/18 , C12N1/19
CPC分类号： C12N15/815 , C07K14/65 , C07K2319/02
摘要： Insulin-like growth factor-1 (IGF-1), a naturally occurring, relatively short, single chain polypeptide, is prepared by growing methylotrophic yeast transformants containing in their genome at least one copy of DNA encoding IGF-1, in operational linkage with DNA encoding a signal sequence, which is effective for directing secretion of proteins from the host cells and which also includes the proteolytic processing site lys-arg and may include one or more glu-ala sequences. In preferred embodiments the signal sequence is the S. cerevisiae alpha mating factor pre-pro sequence. Expression of both the DNA encoding IGF-1 and the pre-pro signal sequence is regulated by a promoter region derived from a methanol responsive gene of a methylotrophic yeast. DNA constructs and recombinant methylotrophic yeast strains used for the expression and secretion of IGF-1 are also provided. For preferred embodiments, protease deficient Pichia pastoris strains are provided.
摘要翻译：胰岛素样生长因子-1（IGF-1）是一种天然存在的相对较短的单链多肽，通过生长含有其基因组的含有编码IGF-1的DNA的至少一个拷贝的甲基营养酵母转化体，与编码信号序列的DNA，其对于引导来自宿主细胞的蛋白质的分泌是有效的，并且还包括蛋白水解加工位点lys-arg，并且可以包括一个或多个葡糖醛酸序列。在优选实施方案中，信号序列是酿酒酵母α交配因子前序序列。编码IGF-1和前一信号序列的DNA的表达由来自甲基营养酵母的甲醇应答基因的启动子区域调节。还提供了用于IGF-1的表达和分泌的DNA构建体和重组甲基营养酵母菌株。对于优选的实施方案，提供了蛋白酶缺乏的巴斯德毕赤酵母菌株。

3. 发明授权

US5324639A Production of insulin-like growth factor-1 in methylotrophic yeast cells 失效
标题翻译：在甲基营养酵母细胞中生产胰岛素样生长因子-1
公开(公告)号：US5324639A
公开(公告)日：1994-06-28
申请号：US023463
申请日：1993-02-25
申请人： Russell A. Brierley , Geneva R. Davis , Gregory C. Holtz
发明人： Russell A. Brierley , Geneva R. Davis , Gregory C. Holtz
IPC分类号： C12N1/19 , C07K14/65 , C12N15/09 , C12N15/18 , C12N15/81 , C12R1/84 , C12P21/06 , C12N1/16 , C12N15/00
CPC分类号： C07K14/65 , C12N15/815 , C07K2319/02
摘要： Insulin-like growth factor-1 (IGF-1), a naturally occurring, relatively short, single chain polypeptide, is prepared by growing methylotrophic yeast transformants containing in their genome at least one copy of a DNA sequence operably encoding IGF-1, in operational association with a DNA sequence encoding the S. cerevisiae alpha mating factor pre-pro sequence (including the proteolytic processing site: lys-arg), both under the regulation of a promoter region of a gene of a methylotrophic yeast, under conditions allowing expression of said DNA sequences, and secretion of IGF-1 into the culture medium.Also disclosed are novel DNA fragments and novel recombinant yeast strains which are useful in the practice of the present invention.
摘要翻译：胰岛素样生长因子-1（IGF-1）是一种天然存在的，相对较短的单链多肽，通过生长甲基营养酵母转化体，其在基因组中含有至少一个拷贝的可操作地编码IGF-1的DNA序列，与编码酿酒酵母α交配因子前序列（包括蛋白水解加工位点：lys-arg）的DNA序列的操作关联，两者都在甲基营养酵母基因的启动子区域的调节下，在允许表达的条件下的所述DNA序列，并将IGF-1分泌到培养基中。还公开了可用于本发明实践的新型DNA片段和新型重组酵母菌株。

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