公开(公告)号：US5198349A

公开(公告)日：1993-03-30

申请号：US707211

申请日：1991-05-23

申请人： Randal J. Kaufman

发明人： Randal J. Kaufman

IPC分类号： A61K38/00 ,  C07K14/755

CPC分类号： C07K14/755 ,  A61K38/00 ,  Y10S435/948 ,  Y10S930/10

摘要： An improved method for producing Factor VIII:c is disclosed. The method involves culturing mammalian cells which contain DNA encoding Factor VIII:c and which are capable of expressing Factor VIII:c. In accordance with this invention the cells are cultured in a medium containing an effective amount of a Factor VIII:c-stabilizing substance comprising (a) von Willebrand Factor (VWF), (b) a phospholipid or phospholipid mixture, or a mixture of (a) and (b).

摘要翻译： 公开了一种用于生产因子VIII：c的改进方法。 该方法包括培养含有编码因子VIII：c并能够表达因子VIII的DNA的哺乳动物细胞：c。 根据本发明，细胞在含有有效量的因子VIII：c稳定物质的培养基中培养，所述因子VIII：c稳定物质包含（a）von Willebrand因子（VWF），（b）磷脂或磷脂混合物，或（ a）和（b）。

公开(公告)号：US4675285A

公开(公告)日：1987-06-23

申请号：US652316

申请日：1984-09-19

申请人： Steven C. Clark ,  Randal J. Kaufman ,  Gordon G. Wong

发明人： Steven C. Clark ,  Randal J. Kaufman ,  Gordon G. Wong

IPC分类号： C07K14/53 ,  C12N15/10 ,  C12N15/85 ,  C12Q1/68 ,  C12N5/00 ,  C12N15/00 ,  C12P21/00

CPC分类号： C12N15/1086 ,  C07K14/53 ,  C12N15/85

摘要： A method for identifying and isolating clones containing DNA coding for a desired protein is described. DNA prepared from a cell that expresses the desired protein is inserted into an isolation expression vector having means for replication (as a means of producing DNA) and a suitable promoter for expression of said DNA in a predetermined mammalian host cell as well as means for replication in a bacterial cell. The transient expression vector is then inserted into a bacterial cell for replication of the DNA. Pools of DNA, prepared from a predetermined number of bacterial clones so that the nucleic acids (DNA and RNA) is substantially free of other bacterial contaminants are transfected or microinjected into mammalian host cells and conditioned medium from growing such cells is tested for the presence of the desired protein. Positive pools are selected and the clones used to make the pool are screened to identify and isolate the clone containing the desired DNA.

摘要翻译： 描述了鉴定和分离含有编码所需蛋白质的DNA的克隆的方法。 将从表达所需蛋白质的细胞制备的DNA插入具有复制方式的分离表达载体（作为生产DNA的手段）和用于在预定哺乳动物宿主细胞中表达所述DNA的合适启动子以及复制手段 在细菌细胞中。 然后将瞬时表达载体插入用于DNA复制的细菌细胞中。 从预定数量的细菌克隆制备DNA，使得核酸（DNA和RNA）基本上不含其它细菌污染物的DNA转染或显微注射到哺乳动物宿主细胞中，并测试来自培养这样的细胞的条件培养基的存在 所需蛋白质。 选择阳性池，筛选用于制备池的克隆以鉴定并分离含有所需DNA的克隆。

公开(公告)号：US20130072434A1

公开(公告)日：2013-03-21

申请号：US13542010

申请日：2012-07-05

申请人： Randal J. Kaufman ,  Steven W. Pipe

发明人： Randal J. Kaufman ,  Steven W. Pipe

IPC分类号： C07K14/755 ,  A61K38/37

CPC分类号： C07K14/755 ,  A61K38/00 ,  A61K38/37

摘要： The present invention provides novel purified and isolated nucleic acid sequences encoding procoagulant-active FVIII proteins. The nucleic acid sequences may encode amino acid sequences corresponding to known human FVIII sequences, wherein residue Phe309 is mutated. The nucleic acid sequences also may encode amino acid sequences corresponding to known human FVIII sequences, wherein the APC cleavage sites, Arg336 and Ile562, are mutated. The nucleic acid sequences of the present invention further encode amino acid sequences corresponding to known human FVIII sequences, wherein the B-domain is deleted, the von Willebrand factor binding site is deleted, a thrombin cleavage site is mutated, an amino acid sequence spacer is inserted between the A2- and A3-domains. Provided herein are methods of producing the FVIII proteins of the invention, nucleotide sequences encoding such proteins, pharmaceutical compositions containing the nucleotide sequences or proteins, as well as methods of treating patients suffering from hemophilia.

摘要翻译： 本发明提供了编码促凝血活性FVIII蛋白的新型纯化和分离的核酸序列。 核酸序列可以编码对应于已知人FVIII序列的氨基酸序列，其中残基Phe309被突变。 核酸序列还可以编码对应于已知人FVIII序列的氨基酸序列，其中APC切割位点Arg336和Ile562被突变。 本发明的核酸序列进一步编码对应于已知人FVIII序列的氨基酸序列，其中缺失B结构域，缺失血管性血友病因子结合位点，突变凝血酶切割位点，氨基酸序列间隔物 插入在A2-和A3-域之间。 本文提供了制备本发明的FVIII蛋白质，编码这种蛋白质的核苷酸序列，含有核苷酸序列或蛋白质的药物组合物以及治疗患有血友病患者的方法。

公开(公告)号：US20120190623A1

公开(公告)日：2012-07-26

申请号：US13015043

申请日：2011-01-27

申请人： Randal J. Kaufman ,  Steven W. Pipe

发明人： Randal J. Kaufman ,  Steven W. Pipe

IPC分类号： A61K38/37 ,  A61K31/7088 ,  C12N5/10 ,  A61P7/04 ,  C12N15/63 ,  C07H21/04 ,  C07K14/755

CPC分类号： C07K14/755 ,  A61K38/00 ,  A61K38/37

摘要： The present invention provides novel purified and isolated nucleic acid sequences encoding procoagulant-active FVIII proteins. The nucleic acid sequences of the present invention encode amino acid sequences corresponding to known human FVIII sequences, wherein residue Phe309 is mutated. The nucleic acid sequences of the present invention also encode amino acid sequences corresponding to known human FVIII sequences, wherein the APC cleavage sites, Arg336 and Ile562, are mutated. The nucleic acid sequences of the present invention further encode amino acid sequences corresponding to known human FVIII sequences, wherein the B-domain is deleted, the von Willebrand factor binding site is deleted, a thrombin cleavage site is mutated, an amino acid sequence spacer is inserted between the A2- and A3-domains. Methods of producing the FVIII proteins of the invention, nucleotide sequences encoding such proteins, pharmaceutical compositions containing the nucleotide sequences or proteins, as well as methods of treating patients suffering from hemophilia, are also provided.

摘要翻译： 本发明提供了编码促凝血活性FVIII蛋白的新型纯化和分离的核酸序列。 本发明的核酸序列编码对应于已知人FVIII序列的氨基酸序列，其中残基Phe309被突变。 本发明的核酸序列还编码对应于已知人FVIII序列的氨基酸序列，其中APC切割位点Arg336和Ile562突变。 本发明的核酸序列进一步编码对应于已知人FVIII序列的氨基酸序列，其中缺失B结构域，缺失血管性血友病因子结合位点，突变凝血酶切割位点，氨基酸序列间隔物 插入在A2-和A3-域之间。 还提供了产生本发明的FVIII蛋白的方法，编码这种蛋白质的核苷酸序列，含有核苷酸序列或蛋白质的药物组合物，以及治疗患有血友病的患者的方法。

公开(公告)号：US5986079A

公开(公告)日：1999-11-16

申请号：US480382

申请日：1995-06-07

申请人： Philip J. Barr ,  Anthony J. Brake ,  Randal J. Kaufman ,  Patricia Tekamp-Olson ,  Louise Wasley ,  Polly A. Wong

发明人： Philip J. Barr ,  Anthony J. Brake ,  Randal J. Kaufman ,  Patricia Tekamp-Olson ,  Louise Wasley ,  Polly A. Wong

IPC分类号： C12N5/10 ,  C07K14/755 ,  C12N9/50 ,  C12N9/64 ,  C12N15/09 ,  C12N15/57 ,  C12P21/02 ,  C12N15/79 ,  C12N15/85

CPC分类号： C12N9/6454 ,  C07K14/755

摘要： Compositions and methods are provided for endopeptidase production and for enhanced efficiencies of processing heterologous precursor polypeptides to mature polypeptides, including proteins requiring gamma-carboxylation for biological activity. These compositions and methods utilize recombinant PACE, a mammalian endopeptidase that is specific for dibasic amino acid sites.

公开(公告)号：US5965425A

公开(公告)日：1999-10-12

申请号：US745880

申请日：1996-11-08

申请人： Philip J. Barr ,  Anthony J. Brake ,  Randal J. Kaufman ,  Patricia Tekamp-Olson ,  Louise Wasley ,  Polly A. Wong

发明人： Philip J. Barr ,  Anthony J. Brake ,  Randal J. Kaufman ,  Patricia Tekamp-Olson ,  Louise Wasley ,  Polly A. Wong

IPC分类号： C12N5/10 ,  C07K14/755 ,  C12N9/50 ,  C12N9/64 ,  C12N15/09 ,  C12N15/57 ,  C12P21/02 ,  C12N9/48 ,  C12N15/79

CPC分类号： C12N9/6454 ,  C07K14/755

摘要： Compositions and methods are provided for endopeptidase production and for enhanced efficiencies of processing heterologous precursor polypeptides to mature polypeptides, including proteins requiring gamma-carboxylation for biological activity. These compositions and methods utilize recombinant PACE, a mammalian endopeptidase that is specific for dibasic amino acid sites.

摘要翻译： 提供用于内肽酶生产的组合物和方法，以及提高加工异源前体多肽对成熟多肽（包括需要γ-羧化生物活性的蛋白质）的效率。 这些组合物和方法利用重组PACE，一种特异于二元氨基酸位点的哺乳动物内肽酶。

公开(公告)号：US5891429A

公开(公告)日：1999-04-06

申请号：US466308

申请日：1995-06-06

申请人： Steven C. Clark ,  Randal J. Kaufman ,  Gordon G. Wong ,  Elizabeth A. Wang

发明人： Steven C. Clark ,  Randal J. Kaufman ,  Gordon G. Wong ,  Elizabeth A. Wang

IPC分类号： A61K38/00 ,  C07K14/535 ,  C12N15/27 ,  C12N15/70 ,  C12N15/81 ,  C12N15/85 ,  A61K38/19 ,  C07K1/20

CPC分类号： C12N15/85 ,  C07K14/535 ,  C12N15/70 ,  C12N15/81 ,  A61K38/00 ,  C12N2800/206 ,  C12N2830/00 ,  C12N2830/702 ,  C12N2840/105 ,  C12N2840/44

摘要： A method for preparing and isolating a transformation vector containing CSF/cDNA is described. The method comprises:preparing RNA from a cell that produces CSF;preparing polyadenylated messenger RNA from said RNA;preparing single stranded cDNA from said messenger RNA;converting the single stranded cDNA to double stranded cDNA;inserting the double stranded cDNA into transformation vectors and transforming bacteria with said vector to form colonies;picking pools of 200 to 500 colonies each and isolating plasmid DNA from each pool;transfecting the plasmid DNA into suitable host cells for expressing CSF protein;culturing the transfected cells and assaying the supernatant for CSF activity; andselecting CSF positive pools and screening the colonies used to make the pool to identify a colony having CSF activity. Also described are a cDNA coding for a protein having CSF activity (i.e. CSF/cDNA), a microorganism or cell line transformed with a recombinant vector containing such CSF/cDNA, and a method for producing CSF protein by expressing said CSF/cDNA by culturing a microorganism or cell line. The invention also provides a method of purifying the CSF proteins and the purified proteins so produced.

摘要翻译： 描述了制备和分离含有CSF / cDNA的转化载体的方法。 该方法包括：从产生CSF的细胞制备RNA; 从所述RNA制备聚腺苷酸化的信使RNA; 从所述信使RNA制备单链cDNA; 将单链cDNA转化为双链cDNA; 将双链cDNA插入转化载体并用所述载体转化细菌以形成菌落; 分别取200至500个菌落的池，每个池中分离出质粒DNA; 将质粒DNA转染到合适的宿主细胞中以表达CSF蛋白; 培养转染的细胞并测定上清液的CSF活性; 并选择CSF阳性池并筛选用于制备池的菌落以鉴定具有CSF活性的菌落。 还描述了编码具有CSF活性的蛋白质（即CSF / cDNA）的cDNA，用含有这种CSF / cDNA的重组载体转化的微生物或细胞系，以及通过培养表达所述CSF / cDNA来产生CSF蛋白的方法 微生物或细胞系。 本发明还提供了纯化CSF蛋白质和如此制备的纯化蛋白质的方法。

公开(公告)号：US5460950A

公开(公告)日：1995-10-24

申请号：US885972

申请日：1992-05-20

申请人： Philip J. Barr ,  Anthony J. Brake ,  Randal J. Kaufman ,  Louise Wasley ,  Patricia Tekamp-Olson ,  Polly A. Wong

发明人： Philip J. Barr ,  Anthony J. Brake ,  Randal J. Kaufman ,  Louise Wasley ,  Patricia Tekamp-Olson ,  Polly A. Wong

IPC分类号： C12N5/10 ,  C07K14/755 ,  C12N9/50 ,  C12N9/64 ,  C12N15/09 ,  C12N15/57 ,  C12P21/02 ,  C12N15/00 ,  C12N15/81 ,  C12N15/85

CPC分类号： C12N9/6454 ,  C07K14/755

摘要： Compositions and methods are provided for endopeptidase production and for enhanced efficiencies of processing heterologous precursor polypeptides to mature polypeptides, including proteins requiring gamma-carboxylation for biological activity. These compositions and methods utilize recombinant PACE, a mammalian endopeptidase that is specific for dibasic amino acid sites.

摘要翻译： 提供用于内肽酶生产的组合物和方法，以及提高加工异源前体多肽对成熟多肽（包括需要γ-羧化生物活性的蛋白质）的效率。 这些组合物和方法利用重组PACE，一种特异于二元氨基酸位点的哺乳动物内肽酶。

公开(公告)号：US5002874A

公开(公告)日：1991-03-26

申请号：US561217

申请日：1990-07-30

申请人： Randal J. Kaufman

发明人： Randal J. Kaufman

IPC分类号： C07K14/47 ,  C12N15/20 ,  C12N15/79

CPC分类号： C07K14/47 ,  C12N15/79

摘要： Eucaryotic host cells are disclosed which contain a DNA molecule encoding an eIF-2.alpha. mutant and preferably a DNA sequence encoding a desired heterologous protein. The DNA sequences are linked to expression control sequences permitting expression of the mutant eIF-2.alpha. gene and the heterologous gene. Culturing such cells provides a method for the production of the desired heterologous protein. The mutations eliminate one or both serine residues at positions 48 and 51 of the eIF-2 sequence. In another aspect of the invention, the eIF-2 5'-untranslated sequence was observed to have effects on translation of heterologous mRNAs.

摘要翻译： 公开了包含编码eIF-2α突变体并优选编码所需异源蛋白质的DNA序列的DNA分子的真核宿主细胞。 DNA序列与允许突变eIF-2α基因和异源基因表达的表达调控序列连接。 培养这样的细胞提供了产生所需异源蛋白质的方法。 突变消除了eIF-2序列的位置48和51处的一个或两个丝氨酸残基。 在本发明的另一方面，观察到eIF-2 5'-非翻译序列对异源mRNA的翻译具有影响。

公开(公告)号：US08597910B1

公开(公告)日：2013-12-03

申请号：US07559509

申请日：1990-07-23

申请人： David Ginsburg ,  Stuart H. Orkin ,  Randal J. Kaufman

发明人： David Ginsburg ,  Stuart H. Orkin ,  Randal J. Kaufman

IPC分类号： C12P21/04 ,  C12N15/00 ,  C12N5/07 ,  A61K35/14 ,  C07H21/04

CPC分类号： C07K14/755 ,  A61K38/37

摘要： Von Willebrand's Factor (VWF) is produced using an expression vector that includes: 1) a DNA sequence encoding a functional VWF protein; and 2) regulatory DNA capable of effecting expression of that DNA sequence in a host cell transformed with the vector. Restriction fragment length polymorphisms (RFLP's) associated with the VWF gene are identified and used in a probe for determining the source of a VWF gene in a DNA sample. The gene for VWF is localized to the short arm of human chromosome 12 (12p).

摘要翻译： 冯维勒布兰氏因子（VWF）使用表达载体产生，所述表达载体包括：1）编码功能性VWF蛋白的DNA序列; 和2）能够在用载体转化的宿主细胞中实现该DNA序列的表达的调节DNA。 鉴定与VWF基因相关的限制性片段长度多态性（RFLP），并用于确定DNA样品中VWF基因来源的探针中。 VWF的基因定位于人染色体12（12p）的短臂。