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1. 发明授权

US09783859B2 Method of screening and quantifying various enzymatic activities using artificial genetic circuits 有权
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.

2. 发明授权

US08647854B2 Metagenome-derived alkaline phosphatase 有权
标题翻译：来自Metagenome的碱性磷酸酶
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。

3. 发明申请

US20130052660A1 METHOD FOR DETECTING PROTEIN-PROTEIN INTERACTIONS IN CELLS 有权
标题翻译：检测细胞中蛋白质 - 蛋白质相互作用的方法
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。

4. 发明授权

US08101355B2 Method for cloning and expressing target gene by homologous recombination 有权
标题翻译：通过同源重组克隆和表达靶基因的方法
公开(公告)号：US08101355B2
公开(公告)日：2012-01-24
申请号：US12559345
申请日：2009-09-14
申请人： Seung-Goo Lee , Jae-Jun Song , Jeong-Min Lee , Jae-Seok Ha
发明人： Seung-Goo Lee , Jae-Jun Song , Jeong-Min Lee , Jae-Seok Ha
IPC分类号： C12Q1/68
CPC分类号： C12N15/70
摘要： A method for cloning and expressing a target gene by homologous recombination, and more particularly a method for cloning and expressing a target gene by homologous recombination, wherein a host cell transformed with a recombinant vector and a plasmid containing a recombinase system is introduced with a linear DNA fragment comprising a target gene and a sequence having homology to the recombinant vector. Because complicated genetic engineering steps, such as the restriction enzyme treatment and ligation of a vector and a target gene, are not required, the cloning of a gene can be performed without needing a high degree of skill, and enzyme cost can be reduced. The inventive method can be effectively used for the massive, high-speed cloning and protein expression of genes, and the disclosed pRMT-iTGR system can be used as an analytical means for improving high-efficiency recombinase.
摘要翻译：通过同源重组克隆和表达靶基因的方法，更具体地，通过同源重组克隆和表达靶基因的方法，其中用重组载体转化的宿主细胞和含有重组酶系统的质粒被引入线性包含靶基因的DNA片段和与重组载体具有同源性的序列。因为不需要复杂的基因工程步骤，例如限制酶处理和载体和靶基因的连接，所以可以在不需要高技能的情况下进行基因的克隆，并且可以降低酶成本。本发明的方法可以有效地用于基因的大量高速克隆和蛋白质表达，并且所公开的pRMT-iTGR系统可以用作改进高效重组酶的分析手段。

5. 发明申请

US20070148775A1 Method for cloning and expressing target gene by homologous recombination 审中-公开
标题翻译：通过同源重组克隆和表达靶基因的方法
公开(公告)号：US20070148775A1
公开(公告)日：2007-06-28
申请号：US11487106
申请日：2006-07-14
申请人： Seung-Goo Lee , Jae-Jun Song , Jeong-Min Lee , Jae-Seok Ha
发明人： Seung-Goo Lee , Jae-Jun Song , Jeong-Min Lee , Jae-Seok Ha
IPC分类号： C12N1/21 , C12N15/74
CPC分类号： C12N15/70
摘要： A method for cloning and expressing a target gene by homologous recombination, and more particularly a method for cloning and expressing a target gene by homologous recombination, wherein a host cell transformed with a recombinant vector and a plasmid containing a recombinase system is introduced with a linear DNA fragment comprising a target gene and a sequence having homology to the recombinant vector. Because complicated genetic engineering steps, such as the restriction enzyme treatment and ligation of a vector and a target gene, are not required, the cloning of a gene can be performed without needing a high degree of skill, and enzyme cost can be reduced. The inventive method can be effectively used for the massive, high-speed cloning and protein expression of genes, and the disclosed pRMT-iTGR system can be used as an analytical means for improving high-efficiency recombinase.
摘要翻译：通过同源重组克隆和表达靶基因的方法，更具体地，通过同源重组克隆和表达靶基因的方法，其中用重组载体转化的宿主细胞和含有重组酶系统的质粒被引入线性包含靶基因的DNA片段和与重组载体具有同源性的序列。因为不需要复杂的基因工程步骤，例如限制酶处理和载体和靶基因的连接，所以可以在不需要高技能的情况下进行基因的克隆，并且可以降低酶成本。本发明的方法可以有效地用于基因的大量高速克隆和蛋白质表达，并且所公开的pRMT-iTGR系统可以用作改进高效重组酶的分析手段。

6. 发明申请

US20070128696A1 Novel promoters and gene expression method by using the promoters 有权
标题翻译：通过使用启动子的新型启动子和基因表达方法
公开(公告)号：US20070128696A1
公开(公告)日：2007-06-07
申请号：US11599475
申请日：2006-11-15
申请人： Moon-Hee Sung , Seung-Goo Lee , Seung-Pyo Hong , Hwa-Jung Seo
发明人： Moon-Hee Sung , Seung-Goo Lee , Seung-Pyo Hong , Hwa-Jung Seo
IPC分类号： C12P21/06 , C12N15/74 , C12N1/21 , C07H21/04
CPC分类号： C07K14/32 , C12N15/70 , C12N15/75
摘要： To provide a promoter, a recombinant DNA, a gene expression vector, an expression vector, and a transformant, which are capable of expressing a gene without inducing gene expression with an inducer; and a method for producing a protein and a kit therefor, which can be operated easily and performed inexpensively by convenient and inexpensive steps. An isolated DNA having the nucleotide sequence of SEQ ID NO: 1 or 2 of Sequence Listing or a fragment thereof, wherein the isolated DNA exhibits a constitutive promoter activity in Escherichia coli or a bacterium belonging to the genus Bacillus; an isolated DNA having a nucleotide sequence of a nucleic acid capable of hybridizing to the above DNA, wherein the isolated DNA exhibits a constitutive promoter activity in Escherichia coli or a bacterium belonging to the genus Bacillus; a recombinant DNA comprising the above DNA and a foreign gene, wherein the foreign gene is operably located; a gene expression vector, at least comprising the above DNA; an expression vector comprising the recombinant DNA; a transformant having the above recombinant DNA, or the above expression vector; a method for producing a protein, characterized by culturing the above transformant, and collecting a protein from the resulting culture; and a kit for producing a protein, at least comprising the above DNA, or the above gene expression vector.
摘要翻译：提供能够表达基因而不诱导基因表达的启动子，重组DNA，基因表达载体，表达载体和转化体; 以及用于制备蛋白质及其试剂盒的方法，其可以容易地操作并且通过方便和便宜的步骤廉价地进行。具有序列表的SEQ ID NO：1或2的核苷酸序列或其片段的分离的DNA，其中所述分离的DNA在大肠杆菌或属于芽孢杆菌属的细菌中显示组成型启动子活性; 具有能够与上述DNA杂交的核酸的核苷酸序列的分离的DNA，其中所述分离的DNA在大肠杆菌或属于芽孢杆菌属的细菌中显示出组成型启动子活性; 包含上述DNA和外来基因的重组DNA，其中所述外源基因可操作地定位; 至少包含上述DNA的基因表达载体; 包含重组DNA的表达载体; 具有上述重组DNA的转化体或上述表达载体; 一种生产蛋白质的方法，其特征在于培养上述转化体，并从得到的培养物中收集蛋白质; 以及用于生产至少包含上述DNA或上述基因表达载体的蛋白质的试剂盒。

7. 发明授权

US08877446B2 Method for detecting protein-protein interactions in cells 有权
标题翻译：检测细胞中蛋白质 - 蛋白质相互作用的方法
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。

8. 发明申请

US20120238470A1 METHOD FOR SCREENING AND QUANTIFYING VARIOUS ENZYME ACTIVITIES USING A GENETIC ENZYME SCREENING SYSTEM 有权
标题翻译：使用遗传酶筛选系统筛选和定量各种酶活性的方法
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。

9. 发明授权

US07323328B2 Obligately symbiotic thermophile Symbiobacterium toebii SC-1 producing thermostable L-tyrosine phenol-lyase and L-tryptophan indole-lyase 有权
标题翻译：共生共生嗜热菌共培养杆菌SC-1产生热稳定性L-酪氨酸苯酚裂解酶和L-色氨酸吲哚裂解酶
公开(公告)号：US07323328B2
公开(公告)日：2008-01-29
申请号：US11186559
申请日：2005-07-21
申请人： Moon-Hee Sung , Seung-Goo Lee , Sung-Keun Rhee , Seung-Pyo Hong , Dae-Joong Kang , Su-Mi Kim , Chul-Joong Kim , Jin-Woo Bae , Che Ok Jeon , Joong-Jae Kim , Kwang Kim , Jae Jun Song
发明人： Moon-Hee Sung , Seung-Goo Lee , Sung-Keun Rhee , Seung-Pyo Hong , Dae-Joong Kang , Su-Mi Kim , Chul-Joong Kim , Jin-Woo Bae , Che Ok Jeon , Joong-Jae Kim , Kwang Kim , Jae Jun Song
IPC分类号： C12N1/20 , C12N9/88 , C12P39/00
CPC分类号： C12R1/01 , C12N9/88 , C12P13/227
摘要： The present invention relates to a novel obligately symbiotic thermophile Symbiobacterium toebii SC-1 (Accession NO: KCTC 0685BP), and a thermostable L-tyrosine phenol-lyase and L-tryptophan indole-lyase produced by Symbiobacterium toebii SC-1. The present invention also relates to a pure culture method of Symbiobacterium toebii SC-1, a method of growth measurement for Symbiobacterium toebii SC-1 and its relative symbiotic bacteria showing low-growth yield using nitrate respiration, and a screening method of relative symbiotic strains of Symbiobacterium toebii SC-1 from the environment using a specific antibody to it. Therefore, the thermostable L-tyrosine phenol-lyase and L-tryptophan indole-lyase produced by Symbiobacterium toebii SC-1 in this invention can provide more stable catalysts in enzymatic biotransformation processes which enzymatically produce valuable medico-amino acids 3,4-dehydroxy-L-phenylalanine (L-DOPA) and L-tryptophan.
摘要翻译：本发明涉及一种新型的共生共生嗜热ile ile i i i i i i i i））i i i i i i i i i i i i i i i i i。。。。。。。。。。。。。。。。。。。。本发明还涉及使用硝酸盐呼吸的共生杆菌SC-1的纯培养方法，使用硝酸盐呼吸显示低生长产量的共生杆菌SC-1及其相对共生菌的生长方法，以及相对共生菌株的筛选方法的来自环境的共表达杆菌SC-1使用特异性抗体。因此，在本发明中由共生杆菌（Toibii）SC-1产生的热稳定性L-酪氨酸苯酚裂解酶和L-色氨酸吲哚裂解酶可以在酶生物转化过程中提供更稳定的催化剂，其中酶促产生有价值的羟基 - 氨基酸3,4-脱羟基 - L-苯丙氨酸（L-DOPA）和L-色氨酸。

10. 发明授权

US07183077B2 Promoters and gene expression method by using the promoters 有权
标题翻译：启动子和使用启动子的基因表达方法
公开(公告)号：US07183077B2
公开(公告)日：2007-02-27
申请号：US10258482
申请日：2001-04-26
申请人： Moon-Hee Sung , Seung-Goo Lee , Seung-Pyo Hong , Hwa-Jung Seo
发明人： Moon-Hee Sung , Seung-Goo Lee , Seung-Pyo Hong , Hwa-Jung Seo
IPC分类号： C12P21/00 , C12N1/21 , C12N15/70 , C12N15/74 , C07H21/04
CPC分类号： C07K14/32 , C12N15/70 , C12N15/75
摘要： To provide a promoter, a recombinant DNA, a gene expression vector, an expression vector, and a transformant, which are capable of expressing a gene without inducing gene expression with an inducer; and a method for producing a protein and a kit therefor, which can be operated easily and performed inexpensively by convenient and inexpensive steps. An isolated DNA having the nucleotide sequence of SEQ ID NO: 1 or 2 of Sequence Listing or a fragment thereof, wherein the isolated DNA exhibits a constitutive promoter activity in Escherichia coli or a bacterium belonging to the genus Bacillus; an isolated DNA having a nucleotide sequence of a nucleic acid capable of hybridizing to the above DNA, wherein the isolated DNA exhibits a constitutive promoter activity in Escherichia coli or a bacterium belonging to the genus Bacillus; a recombinant DNA comprising the above DNA and a foreign gene, wherein the foreign gene is operably located; a gene expression vector, at least comprising the above DNA; an expression vector comprising the recombinant DNA; a transformant having the above recombinant DNA, or the above expression vector; a method for producing a protein, characterized by culturing the above transformant, and collecting a protein from the resulting culture; and a kit for producing a protein, at least comprising the above DNA, or the above gene expression vector.
摘要翻译：提供能够表达基因而不诱导基因表达的启动子，重组DNA，基因表达载体，表达载体和转化体; 以及用于制备蛋白质及其试剂盒的方法，其可以容易地操作并且通过方便和便宜的步骤廉价地进行。具有序列表的SEQ ID NO：1或2的核苷酸序列或其片段的分离的DNA，其中所述分离的DNA在大肠杆菌或属于芽孢杆菌属的细菌中显示组成型启动子活性; 具有能够与上述DNA杂交的核酸的核苷酸序列的分离的DNA，其中所述分离的DNA在大肠杆菌或属于芽孢杆菌属的细菌中显示出组成型启动子活性; 包含上述DNA和外来基因的重组DNA，其中所述外源基因可操作地定位; 至少包含上述DNA的基因表达载体; 包含重组DNA的表达载体; 具有上述重组DNA的转化体或上述表达载体; 一种生产蛋白质的方法，其特征在于培养上述转化体，并从得到的培养物中收集蛋白质; 以及用于生产至少包含上述DNA或上述基因表达载体的蛋白质的试剂盒。

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