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    Plasmids containing lambda Pl promoter, and engineered restriction site for convenient replacement of ribosomal binding site, hosts containing the plasmids and related methods
    3.
    发明公开
    Plasmids containing lambda Pl promoter, and engineered restriction site for convenient replacement of ribosomal binding site, hosts containing the plasmids and related methods 失效
    和λPL含有启动子的质粒，以及经改造的限制性位点便于更换一个核糖体结合位点的，含有主机和相应的方法的质粒。

    公开(公告)号：EP0173280A1

    公开(公告)日：1986-03-05

    申请号：EP85110683.1

    申请日：1985-08-26

    申请人： BIO-TECHNOLOGY GENERAL CORPORATION

    发明人： Aviv, Haim ,  Bartfeld, Daniel ,  Gorecki, Marian ,  Hartman, Jacob R. ,  Kanner, Dov ,  Levanon, Avigdor ,  Locker-Giladi, Hilla ,  Oppenheim, Amos B.

    IPC分类号： C12N15/73 ,  C12P21/02 ,  C12N15/67 ,  C12P19/34

    CPC分类号： C12N9/0089 ,  A23K20/168 ,  A61K38/446 ,  C07K14/61 ,  C07K14/775 ,  C12N15/69 ,  C12N15/73

    摘要： An improved vector which upon introduction into a suitable host containing the thermolabile repressor C 1 renders the host capable of effecting expression of a desired gene. The vector is a double-stranded DNA molecule which includes in 5' to 3' order the following: the promoter and operator P L O L from lambda bacteriophage; the N utilization site; a first restriction enzyme site which follows thereafter; a ribosomal binding site; an ATG Initiation codon or DNA which is converted Into an ATG initiation codon upon insertion of the desired gene into the vector, a second restriction enzme site for inserting the gene in phase with the ATG initiation codon; an origin of replication and either a gene associated with a selectable or identifiable phenotypic trait manifested when the vector is present In the host or a fragment designated ci 434 which includes the gene for the cl 343 repressor protein and its associated promoter and operator. The distance between the 3' end of P L O L and the 5' end of the N utilization site is less than about 80 base pairs. The distance between the 3' end of the N utilization site and the 5' end of the ribosomal binding site is less than about 300 base pairs.
    Vectors of the invention may also include a T,T 2 rRNA transcription termination sequence located 3' of the second restriction enzyme site. The inventive vectors may have origin of replications capable of autonomous production in the host of at least 400 constitutive copies. Plasmids have been constructed from the vectors and used to produce bovine, chicken, porcine and human growth hormones. human apolipoprotein E and human superoxide dismutase and ana- loges thereof In host cells.
    Such superoxide dismutase or analogs thereof may be used to catalyze the reduction of superoxide radicais, reduce reperfusion injury, prolong the survival time of isolated organs and prevent neurological InJury.
    Enzymatically active eucaryotic superoxide dismutase or an analog thereof may also be produced by an improved method of this invention which employs a production medium having a concentration of Cu ++ greater than about about 2 ppm.
    . The invention also concerns improved methods of ro- covering purified enzymatically active eucaryotic superoxide dismutase or analogs thereof.

    摘要翻译： （1）向做了引入合适的细菌宿主细胞contg。 不耐热阻遏C（I）使宿主细胞能，当温度。 被升高到使阻遏物失活 - 影响由该基因编码插入以产生多肽（Ⅰ）载体中的目的基因的表达的，电平是新当其包含双链DNA分子包括在5“到 3“顺序。 （A）的DNA序列contg。 从拉姆达噬菌体的启动子和操纵P（L）O（L）; （B）一个氮利用的网站结合抗终止子N蛋白; （C）的第一限制性内切酶位点允许置换的DNA序列contg的。 核糖体结合位点后，继; （D）的DNA序列contg。 用于制备能够结合在宿主细胞中的核糖体的所需基因的mRNA核糖体结合位点; （E）ATG起始密码子或转化成所希望的基因向载体的插入密码子的DNA序列; 和（f）的第二限制性内切酶位点用于将基因导入与ATG起始密码子相所需载体; 和（g）的DNA序列contg。 从能够在宿主细胞中自主复制的细菌质粒的复制起点; 和选择，DNA序列contg。 与禅意当矢量存在于宿主细胞中，或DNA序列contg可选择或可识别的表型性状相关的基因。 片段C（I）434，包括基因的C（I）434阻遏蛋白和其相关联的启动子和操纵基因序列。

    A method of producing an enzymatically active polypeptide analog of human Cu/Zn SOD
    4.
    发明公开
    A method of producing an enzymatically active polypeptide analog of human Cu/Zn SOD 失效
    生产人类CU / ZN SOD的酶促活性多肽模拟物的方法

    公开(公告)号：EP0483113A3

    公开(公告)日：1992-05-13

    申请号：EP92101250.6

    申请日：1985-08-26

    申请人： BIO-TECHNOLOGY GENERAL CORPORATION

    发明人： Aviv, Haim ,  Bartfeld, Daniel ,  Gorecki, Marian ,  Hartman, Jacob R. ,  Kanner, Dov ,  Levanon, Avigdor ,  Locker-Giladi, Hilla ,  Oppenheim, Amos B.

    IPC分类号： C12N15/53 ,  C12N9/02

    CPC分类号： C12N9/0089 ,  A23K20/168 ,  A61K38/446 ,  C07K14/61 ,  C07K14/775 ,  C12N15/69 ,  C12N15/73

    摘要： An improved vector which upon introduction into a suitable host containing the thermolabile repressor C I renders the host capable of effecting expression of a desired gene. The vector is a double-stranded DNA molecule which includes in 5' to 3' order the following: the promoter and operator P L O L from lambda bacteriophage; the N utilization site; a first restriction enzyme site which follows thereafter; a ribosomal binding site; an ATG initiation codon or DNA which is converted into an ATG initiation codon upon insertion of the desired gene into the vector; a second restriction enzme site for inserting the gene in phase with the ATG initiation codon; an origin of replication and either a gene associated with a selectable or identifiable phenotypic trait manifested when the vector is present in the host or a fragment designated cI⁴³⁴ which includes the gene for the cI⁴³⁴ repressor protein and its associated promoter and operator. The distance between the 3' end of P L O L and the 5' end of the N utilization site is less than about 80 base pairs. The distance between the 3' end of the N utilization site and the 5' end of the ribosomal binding site is less than about 300 base pairs. Vectors of the invention may also include a T₁T₂ rRNA transcription termination sequence located 3' of the second restriction enzyme site. The inventive vectors may have origin of replications capable of autonomous production in the host of at least 400 constitutive copies. Plasmids have been constructed from the vectors and used to produce bovine, chicken, porcine and human growth hormones, human apolipoprotein E and human superoxide dismutase and analogs thereof in host cells. Such superoxide dismutase or analogs thereof may be used to catalyze the reduction of superoxide radicals, reduce reperfusion injury, prolong the survival time of isolated organs and prevent neurological injury. Enzymatically active eucaryotic superoxide dismutase or an analog thereof may also be produced by an improved method of this invention which employs a production medium having a concentration of Cu⁺⁺ greater than about about 2ppm. The invention also concerns improved methods of recovering purified enzymatically active eucaryotic superoxide dismutase or analogs thereof.

    A method of producing an enzymatically active polypeptide analog of human Cu/Zn SOD
    5.
    发明公开
    A method of producing an enzymatically active polypeptide analog of human Cu/Zn SOD 失效
    方法酶催化生物多磷酸盐分解酶Cu / Zn SOD herzustellen。

    公开(公告)号：EP0483113A2

    公开(公告)日：1992-04-29

    申请号：EP92101250.6

    申请日：1985-08-26

    申请人： BIO-TECHNOLOGY GENERAL CORPORATION

    发明人： Aviv, Haim ,  Bartfeld, Daniel ,  Gorecki, Marian ,  Hartman, Jacob R. ,  Kanner, Dov ,  Levanon, Avigdor ,  Locker-Giladi, Hilla ,  Oppenheim, Amos B.

    IPC分类号： C12N15/53 ,  C12N9/02

    CPC分类号： C12N9/0089 ,  A23K20/168 ,  A61K38/446 ,  C07K14/61 ,  C07K14/775 ,  C12N15/69 ,  C12N15/73

    摘要： An improved vector which upon introduction into a suitable host containing the thermolabile repressor C I renders the host capable of effecting expression of a desired gene. The vector is a double-stranded DNA molecule which includes in 5' to 3' order the following: the promoter and operator P L O L from lambda bacteriophage; the N utilization site; a first restriction enzyme site which follows thereafter; a ribosomal binding site; an ATG initiation codon or DNA which is converted into an ATG initiation codon upon insertion of the desired gene into the vector; a second restriction enzme site for inserting the gene in phase with the ATG initiation codon; an origin of replication and either a gene associated with a selectable or identifiable phenotypic trait manifested when the vector is present in the host or a fragment designated cI⁴³⁴ which includes the gene for the cI⁴³⁴ repressor protein and its associated promoter and operator. The distance between the 3' end of P L O L and the 5' end of the N utilization site is less than about 80 base pairs. The distance between the 3' end of the N utilization site and the 5' end of the ribosomal binding site is less than about 300 base pairs.
    Vectors of the invention may also include a T₁T₂ rRNA transcription termination sequence located 3' of the second restriction enzyme site. The inventive vectors may have origin of replications capable of autonomous production in the host of at least 400 constitutive copies.
    Plasmids have been constructed from the vectors and used to produce bovine, chicken, porcine and human growth hormones, human apolipoprotein E and human superoxide dismutase and analogs thereof in host cells.
    Such superoxide dismutase or analogs thereof may be used to catalyze the reduction of superoxide radicals, reduce reperfusion injury, prolong the survival time of isolated organs and prevent neurological injury.
    Enzymatically active eucaryotic superoxide dismutase or an analog thereof may also be produced by an improved method of this invention which employs a production medium having a concentration of Cu⁺⁺ greater than about about 2ppm.
    The invention also concerns improved methods of recovering purified enzymatically active eucaryotic superoxide dismutase or analogs thereof.

    摘要翻译： 一种改进的载体，其在引入含有不耐热阻遏物CI的合适的宿主中时，使宿主能够实现所需基因的表达。 载体是双链DNA分子，其以5'至3'的顺序包括以下：来自λ噬菌体的启动子和操纵子PLOL; N利用场所; 此后第一个限制酶位点; 核糖体结合位点; ATG起始密码子或DNA，将所需基因插入载体后转化为ATG起始密码子; 用于将基因与ATG起始密码子相插入的第二限制性酶切位点; 复制起点和当载体存在于宿主中时表现出的可选择或可识别的表型性状相关的基因或称为cI4 <3>的片段，其包含cI4基因的基因， 3> 4阻抑蛋白及其相关启动子和操纵子。 PLOL的3'端与N使用位点的5'端之间的距离小于约80个碱基对。 N利用位点的3'末端与核糖体结合位点的5'末端之间的距离小于约300个碱基对。 本发明的载体还可以包含位于第二限制酶位点3'的T1T2 rRNA转录终止序列。 本发明的载体可以具有能够在宿主中自主生产至少400种组成型拷贝的重复起源。 已经从载体构建质粒，并用于在宿主细胞中产生牛，鸡，猪和人生长激素，人载脂蛋白E和人超氧化物歧化酶及其类似物。 这种超氧化物歧化酶或其类似物可用于催化超氧自由基的还原，减少再灌注损伤，延长分离的器官的存活时间并预防神经损伤。 酶促活性的真核超氧化物歧化酶或其类似物也可以通过本发明的改进方法生产，其使用浓度大于约2ppm的Cu 2+的生产培养基。 本发明还涉及回收纯化的酶促活性的真核超氧化物歧化酶或其类似物的改进方法。