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1. 发明授权

US5122459A Gene encoding biologically active human interleukin 1 失效
标题翻译：基因编码生物活性人白细胞介素1
公开(公告)号：US5122459A
公开(公告)日：1992-06-16
申请号：US443399
申请日：1989-11-30
申请人： Paul J. Conlon, III , David j. Cosman , Kenneth H. Grabstein , Thomas P. Hopp , Shirley R. Kronheim , Alf D. Larsen , Carl J. March , Virginia L. Price , Douglas P. Cerretti
发明人： Paul J. Conlon, III , David j. Cosman , Kenneth H. Grabstein , Thomas P. Hopp , Shirley R. Kronheim , Alf D. Larsen , Carl J. March , Virginia L. Price , Douglas P. Cerretti
IPC分类号： C07K14/545 , C12N15/25 , C12N15/81
CPC分类号： C07K14/545 , C12N15/81 , Y10S435/942
摘要： Double-stranded cDNA is prepared from polyadenylated RNA extracted from activated human peripheral blood adherent mononuclear cells. The cDNA is inserted within a plasmid vector and then the recombinant plasmid employed to transform an appropriate host. Transformed hosts are identified and grouped into pools. Plasmid DNA prepared from these pools is hybridized with a labeled, synthetic oligonucleotide probe corresponding to a portion of the amino acid sequence of the interleukin 1 protein. Pools of host cells that provide a positive signal to the probe are identified, plated out and then employed in direct bacterial colony hybridization with the same probe, thereby to isolate the particular positive colony. Plasmid DNA is prepared from this colony and characterized by restriction enzyme mapping and sequencing by chain-termination method. The coding region for the IL-1 gene is inserted into a shuttle vector for amplification of the vector followed by expression of functional IL-1.
摘要翻译：双链cDNA由从活化的人外周血粘附单核细胞提取的聚腺苷酸化RNA制备。将cDNA插入质粒载体中，然后将重组质粒用于转化合适的宿主。已识别转化的主机并将其分组到池中。从这些池制备的质粒DNA与对应于白介素1蛋白的氨基酸序列的一部分的标记的合成的寡核苷酸探针杂交。确定提供探针的正信号的宿主细胞池，电镀，然后用于与相同探针的直接细菌菌落杂交，从而分离出特定的阳性菌落。从该菌落制备质粒DNA，并通过限制酶作图和通过链终止法进行测序。将用于IL-1基因的编码区插入穿梭载体中用于扩增载体，随后表达功能性IL-1。

2. 发明授权

US5078996A Activation of macrophage tumoricidal activity by granulocyte-macrophage colony stimulating factor 失效
标题翻译：通过GRANULOCYTE-MACROPHAGE COLONY刺激因子激活大鼠的肿瘤活性
公开(公告)号：US5078996A
公开(公告)日：1992-01-07
申请号：US888995
申请日：1986-07-31
申请人： Paul J. Conlon, III , Kenneth H. Grabstein
发明人： Paul J. Conlon, III , Kenneth H. Grabstein
IPC分类号： A61K35/14 , A61K35/12 , A61K35/26 , A61K35/74 , A61K38/00 , A61K38/16 , A61P35/00 , C07K14/535 , C12N15/00 , C12P21/02
CPC分类号： C07K14/535 , A61K38/00 , Y10S530/828
摘要： Macrophages and precursor monocytes are activated to exhibit tumoricidal activity by stimulation solely with granulocyte-macrophage colony stimulating factor. A patient suffering from tumors can be treated by direct administration of therapeutically effective quantities of activated granulocyte-macrophage colony stimulating factor. Homogeneous granulocyte-macrophage colony stimulating factor for use in activating macrophages and monocyte precursors is prepared by recombinant DNA techniques. The gene coding for granylocyte-macrophage colony stimulating factor is isolated and then recombinant protein product expressed in an appropriate expression system. The granulocyte-macrophage colony stimulating factor recovered from the expression system is purified to homogeneity by reverse phase high-performance liquid chromatography.
摘要翻译：通过仅用粒细胞 - 巨噬细胞集落刺激因子进行刺激，活化巨噬细胞和前体单核细胞以表现出杀肿瘤活性。可以通过直接施用治疗有效量的活化的粒细胞 - 巨噬细胞集落刺激因子来治疗患有肿瘤的患者。通过重组DNA技术制备用于激活巨噬细胞和单核细胞前体的均匀粒细胞 - 巨噬细胞集落刺激因子。分离编码大细胞巨噬细胞集落刺激因子的基因，然后在合适的表达系统中表达重组蛋白产物。从表达系统回收的粒细胞 - 巨噬细胞集落刺激因子通过反相高效液相色谱纯化至均质。

3. 发明授权

US4782137A Synthesis of protein with an identification peptide, and hybrid polypeptide incorporating same 失效
标题翻译：用鉴定肽合成蛋白质，以及掺入其的杂交多肽
公开(公告)号：US4782137A
公开(公告)日：1988-11-01
申请号：US76811
申请日：1987-07-23
申请人： Thomas P. Hopp , Susan L. Bektesh , Paul J. Conlon, III , Carl J. March
发明人： Thomas P. Hopp , Susan L. Bektesh , Paul J. Conlon, III , Carl J. March
IPC分类号： C07K14/55 , C07K16/44 , C12N15/62 , A61K37/02 , C12P21/00
CPC分类号： C07K14/55 , C07K16/44 , C12N15/62 , C07K2319/00 , C07K2319/40 , C07K2319/50
摘要： A hybrid polypeptide composed of an identification peptide and a desired functional protein are produced by recombinant DNA techniques. A DNA expression vector is constructed that includes segments of DNA coding for the identification peptide and the desired functional protein. The identification peptide consists of a highly antigenic N-terminal portion and a C-terminal linking portion that connects the identification peptide to the N-terminal of the functional protein. The linking portion of the identification peptide is cleavable at a specific amino acid residue adjacent the functional protein by use of a sequence specific proteolytic enzyme or chemical proteolytic agent. The hybrid polypeptide expressed by the host cells transformed by the cloning vector is removed therefrom and purfied by affinity chromatography techniques by use of an immobilized ligand specific to the antigenic portion of the identification peptide. The protein is then cleaved from the isolated hybrid polypeptide with an appropriate proteolic enzyme or chemical agent, thereby releasing the mature functional protein in highly purified, highly active state.
摘要翻译：通过重组DNA技术产生由鉴定肽和所需功能性蛋白质组成的杂合多肽。构建DNA表达载体，其包括编码鉴定肽和所需功能性蛋白质的DNA片段。识别肽由高度抗原的N末端部分和连接识别肽与功能性蛋白质的N末端的C-末端连接部分组成。识别肽的连接部分可通过使用序列特异性蛋白水解酶或化学蛋白水解剂在与功能蛋白相邻的特定氨基酸残基处可切割。由克隆载体转化的宿主细胞表达的杂合多肽从其中除去，并通过使用对鉴定肽的抗原部分特异性的固定化配体的亲和层析技术进行纯化。然后用合适的蛋白酶或化学试剂从分离的杂交多肽切割蛋白质，从而释放高度纯化，高活性状态的成熟功能性蛋白质。

4. 发明授权

US5484887A Homogeneous interleukin 1 失效
标题翻译：均匀白细胞介素1
公开(公告)号：US5484887A
公开(公告)日：1996-01-16
申请号：US676533
申请日：1984-11-30
申请人： Shirley R. Kronheim , Carl J. March , Paul J. Conlon, III , Thomas P. Hopp
发明人： Shirley R. Kronheim , Carl J. March , Paul J. Conlon, III , Thomas P. Hopp
IPC分类号： C07K14/545 , C12N15/81 , C07K1/00
CPC分类号： C12N15/81 , C07K14/545 , Y10S930/141
摘要： Interleukin 1 has been purified by use of various techniques including ion exchange chromatography and dye-ligand affinity chromatography. By these techniques, interleukin 1 has been purified to homogeneity. The high purification of interleukin 1 has enabled the amino acid composition of this protein to be ascertained and its amino acid sequence to be partially determined.
摘要翻译：白细胞介素1已经通过使用各种技术纯化，包括离子交换层析和染料 - 配体亲和层析。通过这些技术，白细胞介素1已被纯化至均匀。白细胞介素1的高纯化使得能够确定该蛋白质的氨基酸组成并且其氨基酸序列被部分测定。

5. 发明授权

US4703004A Synthesis of protein with an identification peptide 失效
标题翻译：用鉴定肽合成蛋白质
公开(公告)号：US4703004A
公开(公告)日：1987-10-27
申请号：US573825
申请日：1984-01-24
申请人： Thomas P. Hopp , Susan L. Bektesh , Paul J. Conlon, III , Carl J. March
发明人： Thomas P. Hopp , Susan L. Bektesh , Paul J. Conlon, III , Carl J. March
IPC分类号： G01N33/531 , A61K35/74 , A61K36/06 , A61K38/00 , A61K39/00 , C07C15/04 , C07H21/04 , C07K14/005 , C07K14/195 , C07K14/55 , C07K16/00 , C07K16/44 , C07K19/00 , C12N5/00 , C12N5/10 , C12N15/00 , C12N15/09 , C12N15/62 , C12P21/00 , C12P21/02 , C12P21/08 , C12R1/19 , C12R1/865 , C12R1/91 , G01N33/577 , C12N1/00
CPC分类号： C07K14/55 , C07K16/44 , C12N15/62 , C07K2319/00 , C07K2319/40 , C07K2319/50 , Y10S930/28
摘要： A hybrid polypeptide composed of an identification peptide and a desired functional protein are produced by recombinant DNA techniques. A DNA expression vector is constructed that includes segments of DNA coding for the identification peptide and the desired functional protein. The identification peptide consists of a highly antigenic N-terminal portion and a C-terminal linking portion that connects the identification peptide to the N-terminal of the functional protein. The linking portion of the identification peptide is cleavable at a specific amino acid residue adjacent the functional protein by use of a sequence specific proteolytic enzyme or chemical proteolytic agent. The hybrid polypeptide expressed by the host cells transformed by the cloning vector is removed therefrom and purified by affinity chromatography techniques by use of an immobilized ligand specific to the antigenic portion of the identification peptide. The protein is then cleaved from the isolated hybrid polypeptide with an appropriate proteolic enzyme or chemical agent, thereby releasing the mature functional protein in highly purified, highly active state.
摘要翻译：通过重组DNA技术产生由鉴定肽和所需功能性蛋白质组成的杂合多肽。构建DNA表达载体，其包括编码鉴定肽和所需功能性蛋白质的DNA片段。识别肽由高度抗原的N末端部分和连接识别肽与功能性蛋白质的N末端的C-末端连接部分组成。识别肽的连接部分可通过使用序列特异性蛋白水解酶或化学蛋白水解剂在与功能蛋白相邻的特定氨基酸残基处可切割。由克隆载体转化的宿主细胞表达的杂合多肽从其中除去，并通过亲和层析技术通过使用对鉴定肽的抗原部分特异性的固定化配体进行纯化。然后用合适的蛋白酶或化学试剂从分离的杂交多肽切割蛋白质，从而释放高度纯化，高活性状态的成熟功能性蛋白质。

6. 发明授权

US4658018A Process for producing homogeneous colony stimulating factor 失效
标题翻译：生产均质殖民地刺激因子的方法
公开(公告)号：US4658018A
公开(公告)日：1987-04-14
申请号：US588940
申请日：1984-03-13
申请人： David L. Urdal , Carl J. March , Diane Y. Mochizuki , Paul J. Conlon, III
发明人： David L. Urdal , Carl J. March , Diane Y. Mochizuki , Paul J. Conlon, III
IPC分类号： C07K14/53 , C07K3/20 , C12P21/00
CPC分类号： C07K14/53 , Y10S930/145
摘要： Colony stimulating factor derived from malignant cells has been purified by use of various techniques including multiple high performance liquid chromotography steps. By this technique, colony stimulating factor has been resolved into distinct species, and one of the species denominated as CSF-2A, has been purified to homogeneity. The high purification of the CSF-2A has enabled the amino acid composition of this protein molecule to be partially sequenced with an automated sequencer.
摘要翻译：来自恶性细胞的集落刺激因子已经通过使用包括多个高效液相色谱步骤的各种技术进行了纯化。通过这种技术，集落刺激因子已经被分解成不同的物种，并且被称为CSF-2A的物种之一已经被纯化到同质性。 CSF-2A的高纯化使得该蛋白质分子的氨基酸组成能够用自动测序仪部分测序。

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