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1. 发明授权

US4767708A Enzyme amplification and purification 失效
公开(公告)号：US4767708A
公开(公告)日：1988-08-30
申请号：US638638
申请日：1984-08-07
申请人： Edwin G. Minkley, Jr. , William E. Brown
发明人： Edwin G. Minkley, Jr. , William E. Brown
IPC分类号： C12N9/12 , C12N1/20 , C12N5/00 , C12N15/00
CPC分类号： C12N9/1252
摘要： Restriction enzymes are used to remove from DNA a complete and undamaged structural gene coding region for the expression of DNA polymerase I (polA) without the gene's natural promoter or with only a significantly damaged portion of the gene's natural promoter. Also by the use of restriction enzymes, a segment from a plasmid cloning vector is excised at a position adjacent to a promoter which is conditionally controllable and may be more powerful than the damaged or removed promoter. The gene for DNA polymerase I is enzymatically cloned into said vector at the position of said removed segment and adjacent to said conditionally controllable promoter. Multicopies of the cloned vector are introduced into a host baterial strain (E. coli). The host strain is then cultured so that the cell colony grows and replicates new generations containing replicated foreign plasmid. During such said replication the activity of said controllable promoter is repressed. After the cell colony has grown, the repression of said controllable promoter is removed and the cells express an amplified amount of DNA polymerase I which is lethal or inhibitory to the cells. An improved procedure is disclosed comprising a sequence of steps for harvesting purified DNA polymerase I.

2. 发明授权

US5126270A Enzyme amplification and purification 失效
标题翻译：酶扩增和纯化
公开(公告)号：US5126270A
公开(公告)日：1992-06-30
申请号：US117279
申请日：1987-11-05
申请人： Edwin G. Minkley, Jr.
发明人： Edwin G. Minkley, Jr.
IPC分类号： C12N9/12 , C12N15/54
CPC分类号： C12N9/1252
摘要： Restriction enzymes are used to remove from DNA a complete and undamaged structural gene coding region for the expression of DNA polymerase I (polA) without the gene's natural promoter or with only a significantly damaged portion of the gene's natural promoter. Also by the use of restriction enzymes, a segment from a plasmid cloning vector is excised at a position adjacent to a promoter which is conditionally controllable and may be more powerful than the damaged or removed promoter. The gene for DNA polymerase I is enzymatically cloned into said vector at the position of said removed segment and adjacent to said conditionally controllable promoter. Multicopies of the cloned vector are introduced into a host baterial strain (E. coli). The host strain is then cultured so that the cell colony grows and replicates new generations containing replicated foreign plasmid. During such said replication the activity of said controllable promoter is repressed. After the cell colony has grown, the repression of said controllable promoter is removed and the cells express an amplified amount of DNA polymerase I which is lethal or inhibitory to the cells. An improved procedure is disclosed comprising a sequence of steps for harvesting purified DNA polymerase I.
摘要翻译：使用限制酶从DNA中去除完整和未受损的结构基因编码区，用于表达没有基因天然启动子的DNA聚合酶I（polA）的表达，或仅具有基因天然启动子的显着损伤部分。同样通过使用限制酶，将来自质粒克隆载体的片段在与有条件可控的启动子相邻的位置切除，并且可能比受损或去除的启动子更强大。将DNA聚合酶I的基因酶切地克隆到所述载体中的所述去除区段的位置并与所述条件可控启动子相邻。将克隆载体的多核苷酸引入宿主材料菌株（E.coli）中。然后培养宿主菌株，使得细胞集落生长并复制含有复制的外源质粒的新一代。在所述复制期间，所述可控启动子的活性被抑制。在细胞集落生长之后，去除所述可控启动子的抑制，并且细胞表达对细胞致死或抑制的扩增量的DNA聚合酶I。公开了一种改进的方法，其包括用于收获纯化的DNA聚合酶I的一系列步骤。

3. 发明授权

US6017745A Enzyme amplification and purification process for producing DNA polymerase 失效
标题翻译：用于产生DNA聚合酶的酶扩增和纯化方法
公开(公告)号：US6017745A
公开(公告)日：2000-01-25
申请号：US414133
申请日：1995-03-30
申请人： Edwin G. Minkley, Jr.
发明人： Edwin G. Minkley, Jr.
IPC分类号： C12N9/12 , C12N15/54 , C12N15/70
CPC分类号： C12N9/1252
摘要： Restriction enzymes are used to remove from DNA a complete and undamaged structural gene coding region for the expression of DNA polymerase I (polA) without the gene's natural promoter or with only a significantly damaged portion of the gene's natural promoter. Also by the use of restriction enzymes, a segment from a plasmid cloning vector is excised at a position adjacent to a promoter which is conditionally controllable and may be more powerful than the damaged or removed promoter. The gene for DNA polymerase I is enzymatically cloned into said vector at the position of said removed segment and adjacent to said conditionally controllable promoter. Multicopies of the cloned vector are introduced into a host baterial strain (E. coli). The host strain is then cultured so that the cell colony grows and replicates new generations containing replicated foreign plasmid. During such said replication the activity of said controllable promoter is repressed. After the cell colony has grown, the repression of said controllable promoter is removed and the cells express an amplified amount of DNA polymerase I which is lethal or inhibitory to the cells. An improved procedure is disclosed comprising a sequence of steps for harvesting purified DNA polymerase I.
摘要翻译：使用限制酶从DNA中去除完整和未受损的结构基因编码区，用于表达没有基因天然启动子的DNA聚合酶I（polA）的表达，或仅与基因天然启动子的显着损伤部分。同样通过使用限制酶，将来自质粒克隆载体的片段在与有条件可控的启动子相邻的位置切除，并且可能比受损或去除的启动子更强大。将DNA聚合酶I的基因酶切地克隆到所述载体中的所述去除区段的位置并与所述条件可控启动子相邻。将克隆载体的多核苷酸引入宿主材料菌株（E.coli）中。然后培养宿主菌株，使得细胞集落生长并复制含有复制的外源质粒的新一代。在所述复制期间，所述可控启动子的活性被抑制。在细胞集落生长之后，去除所述可控启动子的抑制，并且细胞表达对细胞致死或抑制的扩增量的DNA聚合酶I。公开了一种改进的方法，其包括用于收获纯化的DNA聚合酶I的一系列步骤。

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