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公开(公告)号：US10301634B2
公开(公告)日：2019-05-28
申请号：US15335132
申请日：2016-10-26
申请人： LONZA LTD.
发明人： Diethard Mattanovich , Brigitte Gasser , Michael Maurer , Roland Prielhofer , Joachim Klein , Jana Wenger
摘要： A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate or at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.
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公开(公告)号：US20140242636A1
公开(公告)日：2014-08-28
申请号：US14350352
申请日：2012-10-05
申请人： LONZA LTD.
发明人： Diethard Mattanovich , Brigitte Gasser , Michael Maurer , Roland Prielhofer , Joachim Klein , Jana Wenger
CPC分类号： C12N15/815 , C07K14/39 , C07K16/00 , C07K2317/55 , C12N9/0008 , C12N15/635 , C12N15/67 , C12N15/81 , C12P21/00 , C12Y102/01012
摘要： A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate of at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.
摘要翻译： 一种通过培养包含表达构建体的重组真核生物细胞系（包括可调节启动子和编码所述启动子的转录控制下的POI的核酸分子）的重组真核生物细胞系的方法，包括以下步骤：a）培养细胞 与抑制启动子的基础碳源连接，b）用限制量的补充碳源培养细胞系，抑制启动子以与天然存在相比以至少15％的转录速率诱导POI的产生 pGAP启动子，和c）生产和回收POI; 并进一步分离可调节的启动子和相应的表达系统。
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公开(公告)号：US11976284B2
公开(公告)日：2024-05-07
申请号：US17698813
申请日：2022-03-18
申请人： LONZA LTD
发明人： Diethard Mattanovich , Brigitte Gasser , Michael Maurer , Roland Prielhofer , Joachim Klein , Jana Wenger
CPC分类号： C12N15/815 , C07K14/39 , C07K16/00 , C12N9/0008 , C12N15/635 , C12N15/67 , C12N15/81 , C12P21/00 , C12Y102/01012 , C07K2317/55
摘要： A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate of at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.
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公开(公告)号：US11168117B2
公开(公告)日：2021-11-09
申请号：US16513022
申请日：2019-07-16
申请人： Lonza LTD
摘要： The invention relates to an isolated nucleic acid sequence comprising a promoter, which is a native sequence of Pichia pastoris comprising the nucleic acid sequence of pCS1 of SEQ ID 1, or a functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID 1, or a combination thereof, expression constructs and recombinant host cells comprising the promoter, and a method of producing a protein of interest under the control of the promoter. It further relates to a method to identify a constitutive promoter from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter which when operatively linked to a nucleotide sequence encoding a protein of interest directs the expression thereof in a host cell at an expression level that is higher than under control of the native pGAP promoter at high and low growth rates.
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公开(公告)号：US10752907B2
公开(公告)日：2020-08-25
申请号：US15750334
申请日：2016-08-05
申请人： LONZA LTD
摘要： An isolated and/or artificial pG1-x promoter, which is a functional variant of the carbon source regulatable pG1 promoter of Pichia pastoris identified by SEQ ID 1, which pG1-x promoter consists of or comprises at least a part of SEQ ID 1 with a length of at least 293 bp, characterized by the following promoter regions: a) at least one core regulatory region comprising the nucleotide sequences SEQ ID 2 and SEQ ID 3; and b) a non-core regulatory region, which is any region within the pG1-x promoter sequence other than the core regulatory region; wherein the pG1-x promoter comprises at least one mutation in any of the promoter regions and a sequence identity of at least 80% in SEQ ID 2 and SEQ ID 3, and a sequence identity of at least 50% in any region other than SEQ ID 2 or SEQ ID 3; and further wherein the pG1-x promoter is characterized by the same or an increased promoter strength and induction ratio as compared to the pG1 promoter, wherein the promoter strength is at least 1.1-fold increased in the induced state as compared to the pG1 promoter, and/or the induction ratio is at least 1.1-fold increased as compared to the pG1 promoter.
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公开(公告)号：US20190337998A1
公开(公告)日：2019-11-07
申请号：US16513022
申请日：2019-07-16
申请人： Lonza Ltd.
摘要： The invention relates to an isolated nucleic acid sequence comprising a promoter, which is a native sequence of Pichia pastoris comprising the nucleic acid sequence of pCS1 of SEQ ID 1, or a functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID 1, or a combination thereof, expression constructs and recombinant host cells comprising the promoter, and a method of producing a protein of interest under the control of the promoter. It further relates to a method to identify a constitutive promoter from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter which when operatively linked to a nucleotide sequence encoding a protein of interest directs the expression thereof in a host cell at an expression level that is higher than under control of the native pGAP promoter at high and low growth rates.
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公开(公告)号：US10428123B2
公开(公告)日：2019-10-01
申请号：US14777400
申请日：2013-12-18
申请人： Lonza Ltd.
摘要： The invention relates to an isolated nucleic acid sequence comprising a promoter, which is a native sequence of Pichia pastoris comprising the nucleic acid sequence of pCS1 of SEQ ID 1, or a functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID 1, or a combination thereof, expression constructs and recombinant host cells comprising the promoter, and a method of producing a protein of interest under the control of the promoter. It further relates to a method to identify a constitutive promoter from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter which when operatively linked to a nucleotide sequence encoding a protein of interest directs the expression thereof in a host cell at an expression level that is higher than under control of the native pGAP promoter at high and low growth rates.
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公开(公告)号：US20160039891A1
公开(公告)日：2016-02-11
申请号：US14777400
申请日：2013-12-18
申请人： LONZA LTD
CPC分类号： C07K14/39 , C12N15/81 , C12N15/815 , C12P21/00
摘要： The invention relates to an isolated nucleic acid sequence comprising a promoter, which is a native sequence of Pichia pastoris comprising the nucleic acid sequence of pCS1 of SEQ ID 1, or a functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID 1, or a combination thereof, expression constructs and recombinant host cells comprising the promoter, and a method of producing a protein of interest under the control of the promoter. It further relates to a method to identify a constitutive promoter from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter which when operatively linked to a nucleotide sequence encoding a protein of interest directs the expression thereof in a host cell at an expression level that is higher than under control of the native pGAP promoter at high and low growth rates.
摘要翻译： 本发明涉及分离的核酸序列，其包含启动子，其是包含SEQ ID No.1的pCS1的核酸序列或其功能活性变体的巴斯德毕赤酵母的天然序列，其是大小变体，突变体或杂种 SEQ ID 1或其组合，表达构建体和包含启动子的重组宿主细胞，以及在启动子控制下产生感兴趣的蛋白质的方法。 它还涉及从真核细胞中鉴定组成型启动子的方法，以及分离的核酸序列，其包含启动子，所述启动子当可操作地连接到编码感兴趣的蛋白质的核苷酸序列时，以其在宿主细胞中的表达水平以 高于天然pGAP启动子在高和低生长速率下的控制。
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公开(公告)号：US20190185866A1
公开(公告)日：2019-06-20
申请号：US16293479
申请日：2019-03-05
申请人： LONZA LTD.
发明人： Diethard Mattanovich , Brigitte Gasser , Michael Maurer , Roland Prielhofer , Joachim Klein , Jana Wenger
CPC分类号： C12N15/815 , C07K14/39 , C07K16/00 , C07K2317/55 , C12N9/0008 , C12N15/635 , C12N15/67 , C12N15/81 , C12P21/00 , C12Y102/01012
摘要： A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate of at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.
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公开(公告)号：US09512432B2
公开(公告)日：2016-12-06
申请号：US14350352
申请日：2012-10-05
申请人： LONZA LTD
发明人： Diethard Mattanovich , Brigitte Gasser , Michael Maurer , Roland Prielhofer , Joachim Klein , Jana Wenger
CPC分类号： C12N15/815 , C07K14/39 , C07K16/00 , C07K2317/55 , C12N9/0008 , C12N15/635 , C12N15/67 , C12N15/81 , C12P21/00 , C12Y102/01012
摘要： A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate of at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.
摘要翻译： 一种通过培养包含表达构建体的重组真核生物细胞系（包括可调节启动子和编码所述启动子的转录控制下的POI的核酸分子）的重组真核生物细胞系的方法，包括以下步骤：a）培养细胞 与抑制启动子的基础碳源连接，b）用限制量的补充碳源培养细胞系，抑制启动子以与天然存在相比以至少15％的转录速率诱导POI的产生 pGAP启动子，和c）生产和回收POI; 并进一步分离可调节的启动子和相应的表达系统。
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