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    Novel cloning vehicles for polypeptide expression in microbial hosts
    1.
    发明授权
    Novel cloning vehicles for polypeptide expression in microbial hosts 失效
    用于微生物宿主中多肽表达的新型克隆载体

    公开(公告)号：US4643969A

    公开(公告)日：1987-02-17

    申请号：US494040

    申请日：1983-07-25

    申请人： Masayori Inouye ,  Yoshihiro Masui

    发明人： Masayori Inouye ,  Yoshihiro Masui

    IPC分类号： C12N15/62 ,  C12N15/70 ,  C12P21/00 ,  C12N1/00 ,  C12N15/00

    CPC分类号： C12N15/62 ,  C12N15/70 ,  Y10S930/30

    摘要： Methods and compositions are provided for regulated expression of polypeptides in transformed bacterial hosts. A novel class of plasmid cloning vehicles includes a DNA sequence coding for the desired polypeptide (on an insertion site therefor) linked for transcriptional expression in reading phase with four functional fragments derived from the lipoprotein gene of E. coli. The plasmids further include a DNA sequence coding for a specific segment of the E. coli lac promoter-operator, which is positioned in the proper orientation for transcriptional expression of the desired polypeptide, as well as a separate functional E. coli lacI gene coding for the associated repressor molecule which can interact with the lac promoter-operator to prevent transcription therefrom. Expression of the desired polypeptide is under the control of both the constitutive promoter and the inducible promoter, although transcription from either promotor is normally blocked by the repressor molecule. However, the repressor can be selectively inactivated by means of an inducer molecule to permit transcriptional expression of the desired polypeptide from both promoter. The methods utilize such plasmids to introduce genetic capability into micro-organisms for the production of proteins, such as medically or commerically useful hormones, enzymes, immunogenic proteins, or intermediates therefor, but only in the presence of an appropriate inducer.

    摘要翻译： 提供了用于在转化的细菌宿主中调节多肽表达的方法和组合物。 一类新颖的质粒克隆载体包括编码与在大肠杆菌的脂蛋白基因中衍生的四个功能片段相连的用于在阅读阶段转录表达的期望多肽（在其插入位点上）的DNA序列。 质粒还包括编码大肠杆菌lac启动子 - 操纵子的特定区段的DNA序列，其定位于所需多肽的转录表达的正确取向，以及编码所述多肽的单独的功能性大肠杆菌lacI基因 可与lac启动子 - 操纵子相互作用以阻止转录的相关阻遏物分子。 所需多肽的表达受组成型启动子和诱导型启动子的控制，尽管来自启动子的转录通常被阻遏物分子阻断。 然而，阻遏物可以通过诱导剂分子选择性地失活以允许来自两个启动子的所需多肽的转录表达。 所述方法利用这种质粒将遗传能力引入微生物以产生蛋白质，例如医学或商业上有用的激素，酶，免疫原性蛋白质或其中间体，但仅在适当的诱导剂存在下。

    Cloning vehicles for polypeptide expression in microbial hosts
    2.
    发明授权
    Cloning vehicles for polypeptide expression in microbial hosts 失效
    克隆载体用于微生物宿主中的多肽表达

    公开(公告)号：US4757013A

    公开(公告)日：1988-07-12

    申请号：US880358

    申请日：1986-06-26

    申请人： Masayori Inouye ,  Yoshihiro Masui

    发明人： Masayori Inouye ,  Yoshihiro Masui

    IPC分类号： C12N15/62 ,  C12N15/70 ,  C12P21/00 ,  C12N1/20 ,  C12N15/00 ,  C12P21/02

    CPC分类号： C12N15/70 ,  C12N15/62 ,  Y10S930/12

    摘要： Methods and compositions are provided for regulated expression and secretion of polypeptides in transformed bacterial hosts. A novel class of plasmid cloning vehicles includes a DNA sequence coding for the desired polypeptide (or an insertion site therefor) linked for transcriptional expression in reading phase with four functional fragments derived from the lipoprotein gene of E. coli. The plasmids also include a DNA fragment coding for the signal peptide of the ompA protein of E. coli, positioned such that the desired polypeptide is expressed with the ompA signal peptide at its amino terminus, thereby allowing efficient secretion across the cytoplasmic membrane. The plasmids further include a DNA sequence coding for a specific segment of the E. coli lac promoter-operator, which is positioned in the proper orientation for transcriptional expression of the desired polypeptide, as well as a separate functional E. coli lacI gene coding for the associated repressor molecule which can interact with the lac promoter-operator to prevent transcription therefrom. Expression of the desired polypeptide is under the control of both the lipoprotein promoter and the lac promoter-operator, although transcription from either promoter is normally blocked by the repressor molecule. However, the repressor can be selectively inactivated by means of an inducer molecule to permit transcriptional expression of the desired polypeptide from both promoters. The methods utilize such plasmids to introduce genetic capability into micro-organisms for the production of normally secreted proteins, such as medically or commercially useful hormones, enzymes, immunogenic proteins, or intermediates therefor, but only in the presence of an appropriate inducer.

    摘要翻译： 提供了用于调节转化细菌宿主中多肽表达和分泌的方法和组合物。 一类新颖的质粒克隆载体包括编码与阅读阶段转录表达连接的期望多肽（或其插入位点）的DNA序列，其中四个功能片段衍生自大肠杆菌的脂蛋白基因。 质粒还包括编码大肠杆菌的ompA蛋白的信号肽的DNA片段，其定位使得所需的多肽在其氨基末端用ompA信号肽表达，从而允许跨细胞质膜的有效分泌。 质粒还包括编码大肠杆菌lac启动子 - 操纵子的特定区段的DNA序列，其定位于所需多肽的转录表达的正确取向，以及编码所述多肽的单独的功能性大肠杆菌lacI基因 可与lac启动子 - 操纵子相互作用以阻止转录的相关阻遏物分子。 所需多肽的表达在脂蛋白启动子和lac启动子 - 操纵子的控制之下，尽管来自任一启动子的转录通常被阻抑物分子阻断。 然而，阻遏物可以通过诱导剂分子选择性地失活以允许来自两个启动子的所需多肽的转录表达。 所述方法利用这种质粒将遗传能力引入微生物以产生正常分泌的蛋白质，例如医学或商业上有用的激素，酶，免疫原性蛋白质或其中间体，但仅在适当的诱导剂存在下。

    Novel cloning vehicles for polypeptide expression in microbial hosts
    3.
    发明授权
    Novel cloning vehicles for polypeptide expression in microbial hosts 失效
    用于微生物宿主中多肽表达的新型克隆载体

    公开(公告)号：US4863855A

    公开(公告)日：1989-09-05

    申请号：US378481

    申请日：1982-05-14

    申请人： Masayori Inouye ,  Kenzo Nakamura ,  Yoshihiro Masui

    发明人： Masayori Inouye ,  Kenzo Nakamura ,  Yoshihiro Masui

    IPC分类号： C12N15/09 ,  C07H21/04 ,  C12N1/20 ,  C12N1/21 ,  C12N15/00 ,  C12N15/62 ,  C12N15/70 ,  C12P21/00

    CPC分类号： C12N15/62 ,  C12N15/70

    摘要： Methods and compositions are provided for regulated expression of polypeptides in transformed bacterial hosts. A novel class of plasmid cloning vehicles includes a DNA sequence coding for the desired polypeptide (or an insertion site therefor) linked for transcriptional expression in reading phase with one or more functional fragments derived from an outer membrane protein gene of a Gram-negative bacterium. The plasmids also include an inducible promoter sequence positioned in the proper orientation for transcriptional expression of the desired polypeptide, as well as a separate DNA sequence coding for a repressor molecule which can interact with the inducible promoter to prevent transcription therefrom. Expression of the desired polypeptide is under the control of both the constitutive promoter and the inducible promoter, although transcription from either promoter is normally blocked by the repressor molecule. However, the repressor can be selectively inactivated by means of an inducer molecule to permit transcriptional expression of the desired polypeptide from both promoters. The methods utilize such plasmids to introduce genetic capability into micro-organisms for the production of proteins, such as medically or commercially useful hormones, enzymes, immunogenic proteins, or intermediates therefor, but only in the presence of an appropriate inducer.

    摘要翻译： 提供了用于在转化的细菌宿主中调节多肽表达的方法和组合物。 一类新颖的质粒克隆载体包括编码所需多肽（或其插入位点）的DNA序列，其与阅读阶段的转录表达连接，其与来自革兰氏阴性细菌的外膜蛋白基因的一种或多种功能片段连接。 质粒还包括位于适当取向的诱导型启动子序列，用于所需多肽的转录表达，以及编码可与诱导型启动子相互作用以阻止其转录的阻遏物分子的单独DNA序列。 所需多肽的表达受组成型启动子和诱导型启动子的控制，尽管来自任一启动子的转录通常被阻遏物分子阻断。 然而，阻遏物可以通过诱导剂分子选择性地失活以允许来自两个启动子的所需多肽的转录表达。 所述方法利用这种质粒将遗传能力引入微生物以产生蛋白质，例如医学或商业上有用的激素，酶，免疫原性蛋白质或其中间体，但仅在适当的诱导剂存在下。

    RNA Interferases and Methods of Use Thereof
    5.
    发明申请
    RNA Interferases and Methods of Use Thereof 审中-公开
    RNA干扰物及其使用方法

    公开(公告)号：US20130071374A1

    公开(公告)日：2013-03-21

    申请号：US13449095

    申请日：2012-04-17

    申请人： Masayori Inouye ,  Junjie Zhang ,  Yong Long Zhang

    发明人： Masayori Inouye ,  Junjie Zhang ,  Yong Long Zhang

    IPC分类号： C12N9/22 ,  C12P19/34 ,  A61K38/46 ,  C12Q1/44

    CPC分类号： C12N9/22 ,  A61K38/465 ,  C12N15/70 ,  C12P19/34 ,  C12Q1/42 ,  C12Q1/44 ,  G01N2333/922 ,  G01N2800/26

    摘要： The present invention is directed to the discovery of a novel family of enzymes designated herein as mRNA interferases that exhibit endoribonuclease activity. The novel finding of the present inventors, therefore, presents new applications for which mRNA interferase nucleic and amino acid sequences, and compositions thereof may be used to advantage. The invention also encompasses screening methods to identify compounds/agents capable of modulating mRNA interferase activity and methods for using such compounds/agents. Also provided is a kit comprising mRNA interferase nucleic and/or amino acid sequences, mRNA interferase activity compatible buffers, and instruction materials.

    摘要翻译： 本发明涉及在本文中称为表达内切核糖核酸酶活性的mRNA干扰酶的新型家族的发现。 因此，本发明人的新发现提出了可以使用mRNA干扰酶核酸和氨基酸序列及其组合物的新用途。 本发明还包括鉴定能够调节mRNA干扰酶活性的化合物/试剂的筛选方法以及使用这些化合物/试剂的方法。 还提供了包含mRNA干扰酶核酸和/或氨基酸序列，mRNA干扰酶活性相容缓冲液和说明材料的试剂盒。

    SINGLE PROTEIN PRODUCTION IN LIVING CELLS FACILITATED BY A MESSENGER RNA INTERFERASE
    7.
    发明申请
    SINGLE PROTEIN PRODUCTION IN LIVING CELLS FACILITATED BY A MESSENGER RNA INTERFERASE 有权
    生物细胞中的单蛋白生产由信使RNA干扰素

    公开(公告)号：US20100035346A1

    公开(公告)日：2010-02-11

    申请号：US11750314

    申请日：2007-05-17

    申请人： Masayori Inouye ,  Junjie Zhang ,  Motoo Suzuki

    发明人： Masayori Inouye ,  Junjie Zhang ,  Motoo Suzuki

    IPC分类号： C12N15/74 ,  C12N1/21

    CPC分类号： C12N9/22 ,  C12N15/67 ,  C12N15/74

    摘要： The present invention describes a single-protein production (SPP) system in living E. coli cells that exploits the unique properties of an mRNA interferase, for example, MazF, a bacterial toxin that is a single stranded RNA- and ACA-specific endoribonuclease, which efficiently and selectively degrades all cellular mRNAs in vivo, resulting in a precipitous drop in total protein synthesis. Concomitant expression of MazF and a target gene engineered to encode an ACA-less mRNA results in sustained and high-level (up to 90%) target expression in the virtual absence of background cellular protein synthesis. Remarkably, target synthesis continues for at least 4 days, indicating that cells retain transcriptional and translational competence despite their growth arrest. SPP technology works well for yeast and human proteins, even a bacterial integral membrane protein. This novel system enables unparalleled signal to noise ratios that should dramatically simplify structural and functional studies of previously intractable but biologically important proteins. The present invention also provides an optimized condensed single protein production system.

    摘要翻译： 本发明描述了利用mRNA干扰酶的独特性质的活的大肠杆菌细胞中的单蛋白质生产（SPP）系统，例如MazF，单链RNA-和ACA特异性内切核糖核酸酶的细菌毒素， 其有效和选择性地降解体内所有细胞mRNA，导致总蛋白质合成的急剧下降。 MazF和旨在编码无ACA mRNA的靶基因的伴随表达导致在虚拟无背景细胞蛋白质合成中的持续和高水平（高达90％）的靶表达。 值得注意的是，目标合成持续至少4天，表明细胞保留转录和翻译能力，尽管其生长停滞。 SPP技术适用于酵母和人类蛋白质，甚至细菌整合膜蛋白。 这种新颖的系统能够实现无与伦比的信噪比，这将显着简化以前难以处理但生物重要的蛋白质的结构和功能研究。 本发明还提供优化的冷凝单蛋白生产系统。

    MULTIFUNCTIONAL BIOSENSOR BASED ON ZNO NANOSTRUCTURES
    8.
    发明申请
    MULTIFUNCTIONAL BIOSENSOR BASED ON ZNO NANOSTRUCTURES 有权
    基于ZNO纳米结构的多功能生物传感器

    公开(公告)号：US20050116263A1

    公开(公告)日：2005-06-02

    申请号：US10456050

    申请日：2003-06-06

    申请人： Yicheng Lu ,  Zheng Zhang ,  Nuri Emanetoglu ,  Masayori Inouye ,  Oleg Mirochnitchenko

    发明人： Yicheng Lu ,  Zheng Zhang ,  Nuri Emanetoglu ,  Masayori Inouye ,  Oleg Mirochnitchenko

    IPC分类号： C12Q1/68 ,  G01N21/64 ,  G01N27/00 ,  G01N27/12 ,  G01N27/414 ,  G01N29/02 ,  G01N33/543 ,  H01L29/84

    CPC分类号： G01N21/6428 ,  C12Q1/6825 ,  C12Q1/6837 ,  G01N27/4145 ,  G01N27/4146 ,  G01N29/022 ,  G01N33/54373 ,  G01N33/5438 ,  G01N2021/6432 ,  G01N2291/011 ,  G01N2291/014 ,  G01N2291/0255 ,  G01N2291/0256 ,  G01N2291/0423 ,  G01N2291/0426 ,  C12Q2565/501 ,  C12Q2565/607

    摘要： The present invention provides the multifunctional biological and biochemical sensor technology based on ZnO nanostructures. The ZnO nanotips serve as strong DNA or protein molecule binding sites to enhance the immobilization. Patterned ZnO nanotips are used to provide conductivity-based biosensors. Patterned ZnO nanotips are also used as the gate for field-effect transistor (FET) type sensors. Patterned ZnO nanotips are integrated with SAW or BAW based biosensors. These ZnO nanotip based devices operate in multimodal operation combining electrical, acoustic and optical sensing mechanisms. The multifunctional biosensors can be arrayed and combined into one biochip, which will enhance the sensitivity and accuracy of biological and biochemical detection due to strong immobilization and multimodal operation capability. Such biological and biochemical sensor technology are useful in detection of RNA-DNA, DNA-DNA, protein-protein, protein-DNA and protein-small molecules interaction. It can be further applied for drug discovery, and for environmental monitoring and protection.

    摘要翻译： 本发明提供了基于ZnO纳米结构的多功能生物和生物化学传感器技术。 ZnO纳米尖端作为强的DNA或蛋白质分子结合位点来增强固定。 图案化的ZnO纳米尖端用于提供基于导电性的生物传感器。 图案化的ZnO纳米技术也用作场效应晶体管（FET）型传感器的栅极。 图案化的ZnO纳米片与SAW或基于BAW的生物传感器集成。 这些基于ZnO纳米管的器件在电气，声学和光学感测机构的多模式操作中工作。 多功能生物传感器可以排列并组合成一个生物芯片，由于强固定和多模态操作能力，提高了生物和生化检测的灵敏度和准确性。 这种生物和生化传感器技术可用于RNA-DNA，DNA-DNA，蛋白质 - 蛋白质，蛋白质 - DNA和蛋白质 - 小分子相互作用的检测。 可进一步应用于药物发现，环境监测和保护。