公开(公告)号：US09783859B2

公开(公告)日：2017-10-10

申请号：US13376783

申请日：2010-06-08

申请人： Seung Goo Lee ,  Eugene Rha ,  Su Lim Choi ,  Jae Jun Song ,  Jong Hyun Choi ,  Hee Sik Kim

发明人： Seung Goo Lee ,  Eugene Rha ,  Su Lim Choi ,  Jae Jun Song ,  Jong Hyun Choi ,  Hee Sik Kim

IPC分类号： C40B30/00 ,  C12Q1/68 ,  C12N15/10

CPC分类号： C12Q1/6897 ,  C12N15/1086

摘要： A method of detecting and quantifying various enzymatic activities using a constructed artificial genetic circuit GESS (genetic enzyme screening system) for sensing phenolic compounds and a method of screening a trace of activities of target enzymes from a metagenome using the artificial genetic circuit, thereby securing target enzyme genes. When the method for screening and quantifying target enzymatic activity is used, useful genes can be screened from various genetic communities, including environmental or metagenomic libraries, at a single cell level in high throughput (million/day). Further, the sensitivity of the genetic circuit to phenol derivatives and the expression thereof can be controlled, and thus the genetic circuit can rapidly sense and quantify various enzymatic activities. Thus, the method can be advantageously used in the protein engineering technology for enzyme modification. Particularly, it can quantitatively investigate enzymatic activity, and thus can be applied to molecular evolution technology.

公开(公告)号：US08647854B2

公开(公告)日：2014-02-11

申请号：US13519013

申请日：2010-06-08

申请人： Seung Goo Lee ,  Su Lim Choi ,  Eugene Rha ,  Jae Jun Song

发明人： Seung Goo Lee ,  Su Lim Choi ,  Eugene Rha ,  Jae Jun Song

IPC分类号： C12N9/16

CPC分类号： C12N9/16

摘要： The present invention relates to a metagenome-derived alkaline phosphatase, and more particularly to a novel, metagenome-derived alkaline phosphatase screened using a artificial genetic circuit that detects phenolic compounds, and a preparation method thereof. A novel alkaline phosphatase according to the present invention has high activity of dephosphorylating DNA and can be inactivated rapidly by simple heat treatment. Thus, it can be used for a dephosphorylation reaction so that genetic manipulations, including genetic cloning, become efficient. In addition, it can be actively expressed in recombinant microorganisms, and thus can be used in various assays, including enzyme immunoassay.

摘要翻译： 本发明涉及一种来源于碱金属蛋白酶的碱性磷酸酶，更具体地说，涉及使用检测酚类化合物的人工遗传回路筛选的新颖的，基因突变型的碱性磷酸酶及其制备方法。 根据本发明的新型碱性磷酸酶具有高的去磷酸化DNA的活性，并且可以通过简单的热处理快速灭活。 因此，它可以用于去磷酸化反应，使得遗传操作（包括遗传克隆）变得有效。 此外，它可以在重组微生物中有效表达，因此可用于各种测定，包括酶免疫测定。

公开(公告)号：US20130052660A1

公开(公告)日：2013-02-28

申请号：US13524868

申请日：2012-06-15

申请人： Seung Goo Lee ,  Su-Lim Choi ,  Jong Sik Gam ,  Jae Jun Song ,  Sang Jun Lee

发明人： Seung Goo Lee ,  Su-Lim Choi ,  Jong Sik Gam ,  Jae Jun Song ,  Sang Jun Lee

IPC分类号： C12P21/02 ,  C12N1/21 ,  C12N5/10 ,  G01N21/64

CPC分类号： G01N21/6428 ,  G01N33/5008 ,  G01N33/542

摘要： The present invention relates to a method for detecting protein-protein interactions in living cells, and more particularly, to a method for providing cells comprising a first construct and a second construct, wherein the first construct comprises a polynucleotide encoding a first fusion protein which comprises a bait protein, a first fluorescent protein and a CBD (cellulose-binding domain) protein, and wherein the second construct comprises a polynucleotide encoding a second fusion protein which comprises a prey protein and a second fluorescent protein so as to allow formation of inclusion bodies, and detecting interactions between the bait protein and the prey protein that are displayed by inclusion bodies, a method for isolating the prey protein bound to the bait protein using the cells comprising the constructs, the cells, and a kit for detecting protein-protein interactions, comprising the constructs.

摘要翻译： 本发明涉及一种用于检测活细胞中蛋白质 - 蛋白质相互作用的方法，更具体地说，涉及提供包含第一构建体和第二构建体的细胞的方法，其中第一构建体包含编码第一融合蛋白的多核苷酸，其包含 诱饵蛋白质，第一荧光蛋白质和CBD（纤维素结合结构域）蛋白质，并且其中第二构建体包含编码第二融合蛋白的多核苷酸，其包含猎物蛋白质和第二荧光蛋白质，以便形成包涵体 并检测由包涵体显示的诱饵蛋白和猎物蛋白之间的相互作用，使用包含构建体的细胞分离与诱饵蛋白结合的猎物蛋白的方法，所述细胞和用于检测蛋白质 - 蛋白质相互作用的试剂盒 ，包括构建体。

公开(公告)号：US20130040366A1

公开(公告)日：2013-02-14

申请号：US13519013

申请日：2010-06-08

申请人： Seung Goo Lee ,  Su Lim Choi ,  Eugene Rha ,  Jae Jun Song

发明人： Seung Goo Lee ,  Su Lim Choi ,  Eugene Rha ,  Jae Jun Song

IPC分类号： C12N9/16 ,  C12N1/15 ,  C12N1/21 ,  C12N1/19 ,  C12N15/55 ,  C12N15/63

CPC分类号： C12N9/16

摘要： The present invention relates to a metagenome-derived alkaline phosphatase, and more particularly to a novel, metagenome-derived alkaline phosphatase screened using a artificial genetic circuit that detects phenolic compounds, and a preparation method thereof. A novel alkaline phosphatase according to the present invention has high activity of dephosphorylating DNA and can be inactivated rapidly by simple heat treatment. Thus, it can be used for a dephosphorylation reaction so that genetic manipulations, including genetic cloning, become efficient. In addition, it can be actively expressed in recombinant microorganisms, and thus can be used in various assays, including enzyme immunoassay.

摘要翻译： 本发明涉及一种来源于碱金属蛋白酶的碱性磷酸酶，更具体地说，涉及使用检测酚类化合物的人工遗传回路筛选的新颖的，基因突变型的碱性磷酸酶及其制备方法。 根据本发明的新型碱性磷酸酶具有高的去磷酸化DNA的活性，并且可以通过简单的热处理快速灭活。 因此，它可以用于去磷酸化反应，使得遗传操作（包括遗传克隆）变得有效。 此外，它可以在重组微生物中有效表达，因此可用于各种测定，包括酶免疫测定。

公开(公告)号：US08877446B2

公开(公告)日：2014-11-04

申请号：US13524868

申请日：2012-06-15

申请人： Seung Goo Lee ,  Su-Lim Choi ,  Jong Sik Gam ,  Jae Jun Song ,  Sang Jun Lee

发明人： Seung Goo Lee ,  Su-Lim Choi ,  Jong Sik Gam ,  Jae Jun Song ,  Sang Jun Lee

IPC分类号： C12Q1/68 ,  G01N33/542 ,  G01N33/50 ,  G01N21/64

CPC分类号： G01N21/6428 ,  G01N33/5008 ,  G01N33/542

摘要： The present invention relates to a method for detecting protein-protein interactions in living cells, and more particularly, to a method for providing cells comprising a first construct and a second construct, wherein the first construct comprises a polynucleotide encoding a first fusion protein which comprises a bait protein, a first fluorescent protein and a CBD (cellulose-binding domain) protein, and wherein the second construct comprises a polynucleotide encoding a second fusion protein which comprises a prey protein and a second fluorescent protein so as to allow formation of inclusion bodies, and detecting interactions between the bait protein and the prey protein that are displayed by inclusion bodies, a method for isolating the prey protein bound to the bait protein using the cells comprising the constructs, the cells, and a kit for detecting protein-protein interactions, comprising the constructs.

摘要翻译： 本发明涉及一种用于检测活细胞中蛋白质 - 蛋白质相互作用的方法，更具体地说，涉及提供包含第一构建体和第二构建体的细胞的方法，其中第一构建体包含编码第一融合蛋白的多核苷酸，其包含 诱饵蛋白质，第一荧光蛋白质和CBD（纤维素结合结构域）蛋白质，并且其中第二构建体包含编码第二融合蛋白的多核苷酸，其包含猎物蛋白质和第二荧光蛋白质，以便形成包涵体 并检测由包涵体显示的诱饵蛋白和猎物蛋白之间的相互作用，使用包含构建体的细胞分离与诱饵蛋白结合的猎物蛋白的方法，所述细胞和用于检测蛋白质 - 蛋白质相互作用的试剂盒 ，包括构建体。

公开(公告)号：US20120238470A1

公开(公告)日：2012-09-20

申请号：US13376783

申请日：2010-06-08

申请人： Seung Goo Lee ,  Eugene Rha ,  Su Lim Choi ,  Jae Jun Song ,  Jong Hyun Choi ,  Hee Sik Kim

发明人： Seung Goo Lee ,  Eugene Rha ,  Su Lim Choi ,  Jae Jun Song ,  Jong Hyun Choi ,  Hee Sik Kim

IPC分类号： C40B30/08

CPC分类号： C12Q1/6897 ,  C12N15/1086

摘要： A method of detecting and quantifying various enzymatic activities using a constructed artificial genetic circuit GESS (genetic enzyme screening system) for sensing phenolic compounds and a method of screening a trace of activities of target enzymes from a metagenome using the artificial genetic circuit, thereby securing target enzyme genes. When the method for screening and quantifying target enzymatic activity is used, useful genes can be screened from various genetic communities, including environmental or metagenomic libraries, at a single cell level in high throughput (million/day). Further, the sensitivity of the genetic circuit to phenol derivatives and the expression thereof can be controlled, and thus the genetic circuit can rapidly sense and quantify various enzymatic activities. Thus, the method can be advantageously used in the protein engineering technology for enzyme modification. Particularly, it can quantitatively investigate enzymatic activity, and thus can be applied to molecular evolution technology.

摘要翻译： 使用用于感测酚类化合物的构建的人工遗传回路GESS（遗传酶筛选系统）检测和定量各种酶活性的方法，以及使用人造遗传回路从宏基因组筛选靶酶的活性痕迹的方法，由此确保靶 酶基因。 当使用筛选和定量靶酶活性的方法时，可以在高通量（百万/天）的单个细胞水平上从各种遗传群落（包括环境或宏基因组文库）筛选有用的基因。 此外，可以控制遗传回路对苯酚衍生物的敏感性及其表达，因此遗传回路可以快速感测和定量各种酶活性。 因此，该方法可有利地用于酶修饰的蛋白质工程技术中。 特别是可以定量研究酶活性，从而可以应用于分子进化技术。