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    SORF CONSTRUCTS AND MULTIPLE GENE EXPRESSION
    2.
    发明申请
    SORF CONSTRUCTS AND MULTIPLE GENE EXPRESSION 审中-公开
    SORF构造和多基因表达

    公开(公告)号：WO2011053699A1

    公开(公告)日：2011-05-05

    申请号：PCT/US2010/054475

    申请日：2010-10-28

    Applicant: ABBOTT LABORATORIES ,  CARSON, Gerald R. ,  GION, Wendy R. ,  KUNES, Yune Z. ,  LEISE III, Walter F. ,  DAVIS-TABER, Rachel A. ,  FUNG, Emma

    Inventor： CARSON, Gerald R. ,  GION, Wendy R. ,  KUNES, Yune Z. ,  LEISE III, Walter F. ,  DAVIS-TABER, Rachel A. ,  FUNG, Emma

    IPC: C07K16/00 ,  C12P21/02 ,  C12P21/06

    CPC classification number: C07K16/00 ,  C07K16/241 ,  C07K16/244 ,  C07K2317/14 ,  C07K2317/21 ,  C07K2317/76 ,  C07K2319/50 ,  C07K2319/92

    Abstract: Embodiments of the invention relate to vector constructs and methods for expression of polypeptides including multimeric products such as therapeutic antibodies. Particular constructs allow for the generation of expression products from a single open reading frame (sORF). An embodiment provides an isolated or purified expression vector for generating one or more recombinant protein products comprising a single open reading frame insert; said insert comprising a signal peptide nucleic acid sequence encoding a signal peptide; a first nucleic acid sequence encoding a first polypeptide; a first intervening nucleic acid sequence encoding a first protein cleavage site, wherein said first protein cleavage site is provided by an intein segment of a Ion protease gene of Pyrococcus or a klbA gene of Pyrococcus or Methanococcus , or a modified intein segment derived therefrom; and a second nucleic acid sequence encoding a second polypeptide. Certain embodiments of constructs and methods employ an intein segment of a Ion protease gene of Pyrococcus abyssi , Pyrococcus furiosus , or Pyrococcus horikoshii OT3; or an intein segment of a klbA gene of Pyrococcus abyssi , Pyrococcus furiosus , or Methanococcus jannaschii ; or other intein segment.

    Abstract translation: 本发明的实施方案涉及用于表达多肽的载体构建体和方法，包括多聚体产物，例如治疗性抗体。 特定结构允许从单个开放阅读框（sORF）生成表达产物。 一个实施方案提供了用于产生一种或多种包含单个开放阅读框插入物的重组蛋白产物的分离或纯化的表达载体; 所述插入片段包含编码信号肽的信号肽核酸序列; 编码第一多肽的第一核酸序列; 编码第一蛋白切割位点的第一插入核酸序列，其中所述第一蛋白切割位点由Pyrococcus的离子蛋白酶基因的内含肽片段或Pyrococcus或Methanococcus的klbA基因或由其衍生的修饰的内含肽片段提供; 和编码第二多肽的第二核酸序列。 构建体和方法的某些实施方案采用激烈的球孢球菌，激烈火球菌或火球菌OT3的离子蛋白酶基因的内含肽片段; 或激烈球菌（Crrococcus abyssi），激烈火球菌（Pyrococcus furiosus）或季戊糖（Methanococcus jannaschii）的klbA基因的内含肽片段; 或其他内含肽片段。

    MULTIPLE GENE EXPRESSION INCLUDING SORF CONSTRUCTS AND METHODS WITH POLYPROTEINS, PRO-PROTEINS, AND PROTEOLYSIS
    4.
    发明申请
    MULTIPLE GENE EXPRESSION INCLUDING SORF CONSTRUCTS AND METHODS WITH POLYPROTEINS, PRO-PROTEINS, AND PROTEOLYSIS 审中-公开
    多种基因表达，包括SORF构建体和方法与聚丙烯酸，蛋白质和蛋白质和蛋白质

    公开(公告)号：WO2007014162A3

    公开(公告)日：2009-05-07

    申请号：PCT/US2006028691

    申请日：2006-07-21

    Applicant: ABBOTT LAB ,  CARSON GERALD R ,  GION WENDY ,  SALFELD JOCHEN G ,  GU JIJIE ,  REGIER DEAN A ,  KUNES YUNE

    Inventor： CARSON GERALD R ,  GION WENDY ,  SALFELD JOCHEN G ,  GU JIJIE ,  REGIER DEAN A ,  KUNES YUNE

    IPC: C12N15/63 ,  C12P21/06 ,  C07H21/02 ,  C07H21/04 ,  C07K16/18 ,  C12N1/18 ,  C12N5/07 ,  C12N5/071 ,  C12N15/00 ,  C12P21/04 ,  C12P21/08

    CPC classification number: C12N15/67 ,  C07K16/00 ,  C07K2319/50 ,  C07K2319/92 ,  C12N15/1055 ,  C12P21/02 ,  C12P21/06

    Abstract: Disclosed are useful constructs and methods for the expression of proteins using primary translation products that are processed within a recombinant host cell. Constructs comprising a single open reading frame (sORF) are described for protein expression including expression of multiple polypeptides. A primary translation product (a pro-protein or a polyprotein) contains polypeptides such as inteins or hedgehog family auto-processing domains, or variants thereof, inserted in frame between multiple protein subunits of interest. The primary product can also contain cleavage sequences such as other proteolytic cleavage or protease recognition sites, or signal peptides which contain recognition sequences for signal peptidases, separating at least two of the multiple protein subunits. The sequences of the inserted auto-processing polypeptides or cleavage sites can be manipulated to enhance the efficiency of expression of the separate multiple protein subunits. Also disclosed are independent aspects of conducting efficient expression, secretion, and/or multimeric assembly of proteins such as immunoglobulins. Where the polyprotein contains immunoglobulin heavy and light chain segments or fragments capable of antigen recognition, in an embodiment a selectable stoichiometric ratio is at least two copies of a light chain segment per heavy chain segment, with the result that the production of properly folded and assembled functional antibody is made. Modified signal peptides, including such from immunoglobulin light chains, are described.

    Abstract translation: 公开了使用在重组宿主细胞内处理的主要翻译产物来表达蛋白质的有用构建体和方法。 描述了包含单个开放阅读框（sORF）的构建体用于蛋白质表达，包括多个多肽的表达。 初级翻译产品（前蛋白质或多蛋白质）含有多肽，例如内含肽或刺猬蛋白家族自身处理结构域或其变体，其插入到感兴趣的多个蛋白质亚基之间。 初级产物还可以含有裂解序列，如其他蛋白水解切割或蛋白酶识别位点，或含有信号肽酶识别序列的信号肽，分离多个蛋白质亚基中的至少两个。 可以操纵插入的自动处理多肽或切割位点的序列，以提高分离的多个蛋白质亚基的表达效率。 还公开了进行蛋白质例如免疫球蛋白的有效表达，分泌和/或多聚体装配的独立方面。 当多蛋白含有免疫球蛋白重链和轻链片段或能够进行抗原识别的片段时，在一个实施方案中，可选择的化学计量比为每个重链段的轻链片段的至少两个拷贝，结果是适当地折叠和组装 制备功能性抗体。 描述了包含免疫球蛋白轻链的修饰的信号肽。

    MULTIPLE GENE EXPRESSION INCLUDING SORF CONSTRUCTS AND METHODS WITH POLYPROTEINS, PRO-PROTEINS, AND PROTEOLYSIS
    5.
    发明申请
    MULTIPLE GENE EXPRESSION INCLUDING SORF CONSTRUCTS AND METHODS WITH POLYPROTEINS, PRO-PROTEINS, AND PROTEOLYSIS 审中-公开
    多种基因表达，包括SORF构建体和方法与聚丙烯酸，蛋白质和蛋白质和蛋白质

    公开(公告)号：WO2007014162A2

    公开(公告)日：2007-02-01

    申请号：PCT/US2006/028691

    申请日：2006-07-21

    Applicant: ABBOTT LABORATORIES ,  CARSON, Gerald R. ,  GION, Wendy ,  SALFELD, Jochen G. ,  GU, Jijie ,  REGIER, Dean A. ,  KUNES, Yune

    Inventor： CARSON, Gerald R. ,  GION, Wendy ,  SALFELD, Jochen G. ,  GU, Jijie ,  REGIER, Dean A. ,  KUNES, Yune

    IPC: C12P21/08 ,  C07H21/04 ,  C12N5/06 ,  C12N5/08 ,  C12N1/18 ,  C07K16/18

    CPC classification number: C12N15/67 ,  C07K16/00 ,  C07K2319/50 ,  C07K2319/92 ,  C12N15/1055 ,  C12P21/02 ,  C12P21/06

    Abstract: Disclosed are useful constructs and methods for the expression of proteins using primary translation products that are processed within a recombinant host cell. Constructs comprising a single open reading frame (sORF) are described for protein expression including expression of multiple polypeptides. A primary translation product (a pro-protein or a polyprotein) contains polypeptides such as inteins or hedgehog family auto-processing domains, or variants thereof, inserted in frame between multiple protein subunits of interest. The primary product can also contain cleavage sequences such as other proteolytic cleavage or protease recognition sites, or signal peptides which contain recognition sequences for signal peptidases, separating at least two of the multiple protein subunits. The sequences of the inserted auto-processing polypeptides or cleavage sites can be manipulated to enhance the efficiency of expression of the separate multiple protein subunits. Also disclosed are independent aspects of conducting efficient expression, secretion, and/or multimeric assembly of proteins such as immunoglobulins. Where the polyprotein contains immunoglobulin heavy and light chain segments or fragments capable of antigen recognition, in an embodiment a selectable stoichiometric ratio is at least two copies of a light chain segment per heavy chain segment, with the result that the production of properly folded and assembled functional antibody is made. Modified signal peptides, including such from immunoglobulin light chains, are described.

    Abstract translation: 公开了使用在重组宿主细胞内处理的主要翻译产物来表达蛋白质的有用构建体和方法。 描述了包含单个开放阅读框（sORF）的构建体用于蛋白质表达，包括多个多肽的表达。 初级翻译产品（前蛋白质或多蛋白质）含有多肽，例如内含肽或刺猬蛋白家族自身处理结构域或其变体，其插入到感兴趣的多个蛋白质亚基之间。 初级产物还可以含有裂解序列，如其他蛋白水解切割或蛋白酶识别位点，或含有信号肽酶识别序列的信号肽，分离多个蛋白质亚基中的至少两个。 可以操纵插入的自动处理多肽或切割位点的序列，以提高分离的多个蛋白质亚基的表达效率。 还公开了进行蛋白质例如免疫球蛋白的有效表达，分泌和/或多聚体装配的独立方面。 当多蛋白含有免疫球蛋白重链和轻链片段或能够进行抗原识别的片段时，在一个实施方案中，可选择的化学计量比为每个重链段的轻链片段的至少两个拷贝，结果是适当地折叠和组装 制备功能性抗体。 描述了包含免疫球蛋白轻链的修饰的信号肽。

    METHODS FOR MONITORING THE STABILITY OF PROTEIN OR CELLULAR PRODUCT SECRETION BY CELLS IN A FERMENTER CULTURE
    6.
    发明申请
    METHODS FOR MONITORING THE STABILITY OF PROTEIN OR CELLULAR PRODUCT SECRETION BY CELLS IN A FERMENTER CULTURE 审中-公开
    用于监测细胞在细胞培养物中蛋白质或细胞产物分泌稳定性的方法

    公开(公告)号：WO9964560A9

    公开(公告)日：2000-04-27

    申请号：PCT/US9912862

    申请日：1999-06-08

    Applicant: BASF AG ,  CARSON GERALD R ,  HAMMILL LINDA

    Inventor： CARSON GERALD R ,  HAMMILL LINDA

    IPC: C12M1/34 ,  G01N33/68 ,  C12N11/04

    CPC classification number: G01N33/68

    Abstract: The invention features a method for monitoring the stability of a protein or other cellular product secreted by cells in a fermenter culture. The method involves, harvesting from a fermenter culture a first sample of cells that secrete a protein or cellular product and incorporating the cells from the first sample into gel microdrops (GMDs), and measuring secretion of the protein or cellular product by cells in one or more GMD samples over time. The protein secreted by the cells may be, for example, an antibody, cytokine, or growth factor. The cellular product secreted by the cells may be, for example, an antibiotic, amino acid, vitamin, or carbohydrate.

    Abstract translation: 本发明的特征在于监测发酵罐培养物中由细胞分泌的蛋白质或其它细胞产物的稳定性的方法。 该方法涉及从发酵罐培养物收获分泌蛋白质或细胞产物的第一个细胞样品，并将来自第一个样品的细胞并入凝胶微滴（GMD）中，并测量细胞在一个或多个细胞中的分泌， 随着时间的推移，更多的GMD样品。 由细胞分泌的蛋白质可以是例如抗体，细胞因子或生长因子。 由细胞分泌的细胞产物可以是例如抗生素，氨基酸，维生素或碳水化合物。

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