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1. 发明授权

US5032510A Method for expression and secretion in bacillus 失效
标题翻译：杆菌中表达和分泌的方法
公开(公告)号：US5032510A
公开(公告)日：1991-07-16
申请号：US210151
申请日：1988-06-17
申请人： Steven Kovacevic , James R. Miller
发明人： Steven Kovacevic , James R. Miller
IPC分类号： C07K14/62 , C12N1/21 , C12N9/22 , C12N15/62 , C12N15/75
CPC分类号： C12N15/75 , C07K14/62 , C12N15/625 , C12N9/22 , C07K2319/02 , C07K2319/036 , C07K2319/75
摘要： The present invention discloses a novel method for expressing and secreting a functional polypeptide in Bacillus including the vectors and transformants for the practice thereof. The vectors comprise the transcriptional and translational activity sequence, and optionally the signal peptide coding sequence, of the Staphylococcus aureus nuclease gene. The vectors and transformants are useful for the production and secretion of human hormones such as human proinsulin.
摘要翻译：本发明公开了一种用于在芽孢杆菌中表达和分泌功能性多肽的新方法，包括用于实施它们的载体和转化体。载体包含金黄色葡萄球菌核酸酶基因的转录和翻译活性序列，以及任选的信号肽编码序列。载体和转化体可用于人类激素（如人胰岛素原）的产生和分泌。

2. 发明授权

US4783405A Recombinant DNA expression vectors useful in bacillus and other host cells 失效
标题翻译：用于杆菌和其他宿主细胞的重组DNA表达载体
公开(公告)号：US4783405A
公开(公告)日：1988-11-08
申请号：US654437
申请日：1984-09-26
申请人： Steven Kovacevic , James R. Miller , Hansen M. Hsiung
发明人： Steven Kovacevic , James R. Miller , Hansen M. Hsiung
IPC分类号： C12N15/75 , C12N15/76 , C12P21/00 , C07H21/00 , C12N1/00 , C12N1/20 , C12N15/00
CPC分类号： C12N15/75 , C12N15/76
摘要： Recombinant DNA expression vectors for use in Bacillus and other host cells are disclosed. The vectors comprise the veg promoter sequence of Bacillus subtilis, a novel ribosome binding site-containing sequence and a sequence that codes for a functional polypeptide. The ribosome binding site-containing sequence is synthesized in accordance with conventional procedures while the veg promoter sequence can be obtained from E. coli K12 JA221/pMS480 (NRRL B-15258). Various sequences that codes for functional polypeptides and method for their expression in Bacillus are also disclosed.
摘要翻译：公开了用于芽孢杆菌属和其他宿主细胞的重组DNA表达载体。载体包含枯草芽孢杆菌的veg启动子序列，新的含核糖体结合位点的序列和编码功能性多肽的序列。含有核糖体结合位点的序列按照常规方法合成，而veg启动子序列可从大肠杆菌K12 JA221 / pMS480（NRRL B-15258）获得。还公开了编码功能性多肽的各种序列及其在芽孢杆菌中表达的方法。

3. 发明授权

US4559300A Method for using an homologous bacillus promoter and associated natural or modified ribosome binding site-containing DNA sequence in streptomyces 失效
标题翻译：在链霉菌中使用同源杆菌启动子和相关天然或修饰的核糖体结合位点的DNA序列的方法
公开(公告)号：US4559300A
公开(公告)日：1985-12-17
申请号：US458803
申请日：1983-01-18
申请人： Steven Kovacevic , Jeffrey T. Fayerman , James R. Miller , Mark A. Richardson
发明人： Steven Kovacevic , Jeffrey T. Fayerman , James R. Miller , Mark A. Richardson
IPC分类号： C12N15/09 , C12N1/00 , C12N15/74 , C12N15/76 , C12P21/00 , C12N1/20 , C12N15/00
CPC分类号： C12N15/74 , C12N15/76 , Y10S435/886
摘要： A method for expressing a functional polypeptide in Streptomyces comprises transforming a Streptomyces host cell with a recombinant DNA expression vector and then culturing the transformed cell under conditions suitable for cell growth. The recombinant DNA expression vector comprises the veg or any other homologous Bacillus promoter, a naturally occurring or modified ribosome binding site-containing DNA sequence and a gene that codes for a functional polypeptide such as human pre-proinsulin. The method is specifically exemplified by use of expression plasmids pOW529, pOW539 and transformants, Streptomyces ambofaciens/pOW529 and Streptomyces ambofaciens/pOW539. The method is broadly applicable and is particularly useful in economically important Streptomyces taxa.
摘要翻译：在链霉菌中表达功能性多肽的方法包括用重组DNA表达载体转化链霉菌宿主细胞，然后在适合于细胞生长的条件下培养转化的细胞。重组DNA表达载体包含veg或任何其他同源芽孢杆菌启动子，天然存在或修饰的含核糖体结合位点的DNA序列和编码诸如人前胰岛素原的功能性多肽的基因。该方法通过使用表达质粒pOW529，pOW539和转化体，淀粉链霉菌（Streptomyces ambofaciens）/ pOW529和淀粉链霉菌（Stubtomyces ambofaciens）/ pOW539来具体举例说明。该方法广泛适用，在经济上重要的链霉菌属分类群中特别有用。

4. 发明授权

US4977089A Vector comprising signal peptide-encoding DNA for use in Bacillus and other microorganisms 失效
标题翻译：包含用于芽孢杆菌和其他微生物的信号肽编码DNA的载体
公开(公告)号：US4977089A
公开(公告)日：1990-12-11
申请号：US10245
申请日：1987-01-30
申请人： Steven Kovacevic , James R. Miller , Loraine E. Veal , John S. Wood
发明人： Steven Kovacevic , James R. Miller , Loraine E. Veal , John S. Wood
IPC分类号： C07K14/61 , C07K14/62 , C12N9/22 , C12N15/75
CPC分类号： C12N15/75 , C07K14/61 , C07K14/62 , C12N9/22 , C07K2319/00 , C07K2319/02 , C07K2319/036 , C07K2319/75
摘要： A novel promoter, ribosome-binding site-encoding DNA, and a number of signal peptide-encoding DNA sequences are described for use in Bacillus and other host cells. The compounds are useful in constructing expression vectors for use in Bacillus and other host cells and drive expression and secretion of proteins such as nuclease and human growth hormone. In addition, a general method for obtaining cells with increased abilities to express and secrete heterologous gene products is described.
摘要翻译：描述了用于芽孢杆菌和其他宿主细胞的新型启动子，核糖体结合位点编码DNA和许多信号肽编码DNA序列。这些化合物可用于构建用于芽孢杆菌属和其他宿主细胞的表达载体，并驱使蛋白质如核酸酶和人生长激素的表达和分泌。另外，描述了获得增加表达和分泌异源基因产物的能力的细胞的一般方法。

5. 发明授权

US5814503A Fusion proteins comprising cell cycle regulatory proteins 失效
标题翻译：包含细胞周期调节蛋白的融合蛋白
公开(公告)号：US5814503A
公开(公告)日：1998-09-29
申请号：US770761
申请日：1996-12-19
申请人： Steven Kovacevic , Keith A. Otto , Ramachandra N. Rao
发明人： Steven Kovacevic , Keith A. Otto , Ramachandra N. Rao
IPC分类号： C07K14/47 , C12N9/12 , C12N15/52
CPC分类号： C12N9/1205 , C07K14/4738
摘要： The present invention provides novel fusion proteins comprising cyclins and CDKs. A preferred embodiment of the invention provides fusion proteins comprising human cyclin D1 and human CDK4. The fusion proteins of the invention optionally contain modifications, which facilitate their purification. Addition of histidine residues to selected constructs allows purification via immobilized metal affinity chromatography. Antigenic determinants allowing monoclonal antibody-based affinity chromatography purification are provided in selected embodiments of the invention. Protease cleavage sites are incorporated in selected constructs to allow cleavage of the regions incorporated in the cyclin-CDK fusion proteins for purification. Additional modifications which facilitate purification include strepavadin binding domains and antigenic determinants for antibody affinity chromatography.
摘要翻译：本发明提供了包含细胞周期蛋白和CDK的新型融合蛋白。本发明的优选实施方案提供包含人细胞周期蛋白D1和人CDK4的融合蛋白。本发明的融合蛋白任选地含有有助于其纯化的修饰。将组氨酸残基添加到选择的构建体允许通过固定的金属亲和层析进行纯化。在本发明的选定实施方案中提供允许基于单克隆抗体的亲和层析纯化的抗原决定簇。将蛋白酶切割位点并入选择的构建体中以允许切割结合在细胞周期蛋白-CDK融合蛋白中的区域进行纯化。促进纯化的其它修饰包括用于抗体亲和层析的链脲霉素结合结构域和抗原决定簇。

6. 发明授权

US4503155A Multifunctional, cloning vectors for use in Streptomyces, Bacillus, and E. coli 失效
标题翻译：用于链霉菌，芽孢杆菌和大肠杆菌+ B的多功能克隆载体
公开(公告)号：US4503155A
公开(公告)日：1985-03-05
申请号：US344710
申请日：1982-02-01
申请人： James R. Miller , Jeffrey T. Fayerman , Steven Kovacevic , Nancy E. Beerman
发明人： James R. Miller , Jeffrey T. Fayerman , Steven Kovacevic , Nancy E. Beerman
IPC分类号： C12N15/09 , C07H21/04 , C12N1/00 , C12N15/75 , C12N15/76 , C12P19/34 , C12R1/07 , C12R1/125 , C12R1/19 , C12R1/465 , C12N15/00 , C12N1/20
CPC分类号： C12N15/75 , C12N15/76 , Y10S435/832 , Y10S435/849 , Y10S435/886
摘要： The present invention discloses multifunctional recombinant DNA cloning vectors for use in Streptomyces, Bacillus, and E. coli. The invention further discloses transformants of the aforementioned vectors.
摘要翻译：本发明公开了用于链霉菌，芽孢杆菌和大肠杆菌的多功能重组DNA克隆载体。本发明还公开了上述载体的转化体。

7. 发明授权

US5070020A Recombinant DNA expression vectors and DNA compounds that encode deacetoxycephalosporin C synthetase 失效
标题翻译：重组DNA表达载体和编码脱乙酰壳多糖C合成酶的DNA化合物
公开(公告)号：US5070020A
公开(公告)日：1991-12-03
申请号：US192273
申请日：1988-05-09
申请人： Thomas D. Ingolia , Steven Kovacevic , James R. Miller , Paul L. Skatrud
发明人： Thomas D. Ingolia , Steven Kovacevic , James R. Miller , Paul L. Skatrud
IPC分类号： C12N1/15 , C12N1/21 , C12N9/02 , C12R1/19 , C12R1/465 , C12R1/745 , C12R1/82
CPC分类号： C12N9/0071
摘要： The present invention provides DNA compounds that encode deacetoxycephalosporin C synthetase (DAOCS) activity. The compounds can be used to construct recombinant DNA expression vectors for a wide variety of host cells, including E. coli, Penicillium, Streptomyces, Aspergillus, and Cephalosporium.

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