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公开(公告)号：US09434774B2
公开(公告)日：2016-09-06
申请号：US13341231
申请日：2011-12-30
IPC分类号： C07K17/00 , C07K14/435 , C12N9/00
CPC分类号： C07K14/43595 , C12N9/93 , C12Y603/02003
摘要： Aggregation is a major cause of the misbehavior of proteins. A system for modifying a protein to create a more stable variant is provided. The method involves identifying non-conserved hydrophobic amino acid residues on the surface of a protein, suitable for mutating to more hydrophilic residues (e.g., charged amino acids). Any number of residues on the surface may be changed to create a variant that is more soluble, resistant to aggregation, has a greater ability to re-fold, and/or is more stable under a variety of conditions. The invention also provides GFP, streptavidin, and GST variants with an increased theoretical net charge created by the inventive technology. Kits are also provided for carrying out such modifications on any protein of interest.
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公开(公告)号：US09200045B2
公开(公告)日：2015-12-01
申请号：US14004280
申请日：2012-03-09
申请人： David R. Liu , Sun H. Peck
发明人： David R. Liu , Sun H. Peck
CPC分类号： C07K14/721 , C07K14/195 , C07K14/35 , C07K14/47 , C07K2319/33 , C07K2319/50 , C07K2319/60 , C07K2319/92 , C12N9/52
摘要： Elucidating the function of proteins in mammalian cells is particularly challenging due to the inherent complexity of these systems. Methods to study protein function in living cells ideally perturb the activity of only the protein of interest but otherwise maintain the natural state of the host cell or organism. Ligand-dependent inteins offer single-protein specificity and other desirable features as an approach to control protein function in cells post-translationally. Some aspects of this invention provide second-generation ligand-dependent inteins that splice to substantially higher yields and with faster kinetics in the presence of the cell-permeable small molecule 4-HT, especially at 37° C., while exhibiting comparable or improved low levels of background splicing in the absence of 4-HT, as compared to the parental inteins. These improvements were observed in four protein contexts tested in mammalian cells at 37° C., as well as in yeast cells assayed at 30° C. or 37° C. The newly evolved inteins described herein are therefore promising tools as conditional modulators of protein structure and function in yeast and mammalian cells.
摘要翻译： 由于这些系统的固有复杂性，阐明了哺乳动物细胞中蛋白质的功能是特别具有挑战性的。 在活细胞中研究蛋白质功能的方法理想地扰乱了仅感兴趣的蛋白质的活性，但是另外保持宿主细胞或生物体的天然状态。 依赖于配体的内含肽提供单蛋白特异性和其他所需特征，作为控制翻译后细胞蛋白质功能的方法。 本发明的一些方面提供第二代配体依赖的内含肽，其在细胞可渗透的小分子4-HT的存在下，特别是在37℃下，以更高的产量和更快的动力学剪接，同时具有相当或改进的低 与亲本内含肽相比，不存在4-HT时的背景剪接水平。 在37℃下在哺乳动物细胞中测试的四种蛋白质上下文以及在30℃或37℃下测定的酵母细胞中观察到这些改善。因此，本文所述的新进化的内含肽是有前途的工具，作为蛋白质的条件调节剂 结构和功能在酵母和哺乳动物细胞。
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公开(公告)号：US09023594B2
公开(公告)日：2015-05-05
申请号：US13062098
申请日：2009-09-08
申请人： David R. Liu , Kevin M. Esvelt
发明人： David R. Liu , Kevin M. Esvelt
CPC分类号： C12N15/1058 , C12N7/00 , C12N15/1024 , C12N15/1037
摘要： The present invention discloses generalizable methods of evolving nucleic acids and proteins utilizing continuous directed evolution. The invention discloses methods of passing a nucleic acid from cell to cell in a desired function-dependent manner. The linkage of the desired function and passage of the nucleic acid from cell to cell allows for continuous selection and mutation of the nucleic acid.
摘要翻译： 本发明公开了利用连续定向进化进化的核酸和蛋白质的可概括的方法。 本发明公开了以期望的功能依赖性方式将核酸从细胞传递到细胞的方法。 所需功能的连接和核酸从细胞到细胞的通过允许核酸的连续选择和突变。
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公开(公告)号：US20120129759A1
公开(公告)日：2012-05-24
申请号：US13341231
申请日：2011-12-30
CPC分类号： C07K14/43595 , C12N9/93 , C12Y603/02003
摘要： Aggregation is a major cause of the misbehavior of proteins. A system for modifying a protein to create a more stable variant is provided. The method involves identifying non-conserved hydrophobic amino acid residues on the surface of a protein, suitable for mutating to more hydrophilic residues (e.g., charged amino acids). Any number of residues on the surface may be changed to create a variant that is more soluble, resistant to aggregation, has a greater ability to re-fold, and/or is more stable under a variety of conditions. The invention also provides GFP, streptavidin, and GST variants with an increased theoretical net charge created by the inventive technology. Kits are also provided for carrying out such modifications on any protein of interest.
摘要翻译： 聚集是蛋白质不正当行为的主要原因。 提供了用于修饰蛋白质以产生更稳定变体的系统。 该方法涉及鉴定蛋白质表面上的非保守疏水性氨基酸残基，适合于突变为更亲水残基（例如带电氨基酸）。 可以改变表面上的任何数量的残基，以产生更可溶，耐聚集，更大的再折叠能力和/或在各种条件下更稳定的变体。 本发明还提供具有由本发明技术产生的增加的理论净电荷的GFP，链霉抗生物素蛋白和GST变体。 还提供了用于对任何感兴趣的蛋白进行这些修饰的试剂盒。
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公开(公告)号：US20110190141A1
公开(公告)日：2011-08-04
申请号：US12834072
申请日：2010-07-12
申请人： David R. Liu , Zev J. Gartner , Jeffrey B. Doyon , Christopher T. Calderone , Matthew W. Kanan , Xiaoyu Li , Thomas M. Snyder , Daniel M. Rosenbaum
发明人： David R. Liu , Zev J. Gartner , Jeffrey B. Doyon , Christopher T. Calderone , Matthew W. Kanan , Xiaoyu Li , Thomas M. Snyder , Daniel M. Rosenbaum
IPC分类号： C40B10/00
CPC分类号： C12Q1/68 , C12N15/1068
摘要： Nature evolves biological molecules such as proteins through iterated rounds of diversification, selection, and amplification. The power of Nature and the flexibility of organic synthesis are combined in nucleic acid-templated synthesis. The present invention provides a variety of template architectures for performing nucleic acid-templated synthesis, methods for increasing the selectivity of nucleic acid-templated reactions, methods for performing stereoselective nucleic acid-templated reactions, methods of selecting for reaction products resulting from nucleic acid-templated synthesis, and methods of identifying new chemical reactions based on nucleic acid-templated synthesis.
摘要翻译： 自然会通过迭代多样化，选择和扩增进化生物分子，如蛋白质。 自然的力量和有机合成的灵活性结合在核酸模板合成中。 本发明提供用于进行核酸模板合成的多种模板结构，用于增加核酸模板化反应的选择性的方法，用于进行立体选择性核酸模板化反应的方法，选择由核酸 - 模板合成，以及基于核酸模板合成鉴定新化学反应的方法。
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公开(公告)号：US20110112040A1
公开(公告)日：2011-05-12
申请号：US12989829
申请日：2009-04-28
CPC分类号： A61K38/17
摘要： Compositions, systems and related methods for delivering a supercharged protein or a complex of a supercharged protein and therapeutic agent (e g, nucleic acid, peptide, small molecule) to cells are disclosed. Superpositively charged proteins may be associated with nucleic acids (which typically have a net negative charge) via electrostatic interactions. The systems and methods may involve altering the primary sequence of a protein in order to “supercharge” the protein (e g, to generate a superpositively-charged protein). The compositions may be used to treat proliferative diseases, infectious diseases, cardiovascular diseases, inborn errors in metabolism, genetic diseases, etc.
摘要翻译： 公开了用于将超荷电蛋白或超荷电蛋白和治疗剂（例如，核酸，肽，小分子）的复合物递送至细胞的组合物，系统和相关方法。 带正电荷的蛋白质可能通过静电相互作用与核酸（通常具有净负电荷）相关联。 所述系统和方法可以涉及改变蛋白质的一级序列以便“增加”蛋白质（例如产生超正电荷的蛋白质）。 该组合物可用于治疗增殖性疾病，感染性疾病，心血管疾病，代谢中的先天错误，遗传疾病等。
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公开(公告)号：US07807408B2
公开(公告)日：2010-10-05
申请号：US11107335
申请日：2005-04-15
申请人： David R. Liu , Joshua A. Bittker , Jane M. Liu
发明人： David R. Liu , Joshua A. Bittker , Jane M. Liu
CPC分类号： C12N15/62 , C12N15/1027 , C12N15/1058 , C12Q1/6806 , C12Q2533/107 , C12Q2525/203 , C12Q2521/301
摘要： Disclosed is a method of altering a nucleic acid such as RNA or DNA. The method comprises fragmenting a parent nucleic acid strand to generate nucleic acid fragments. At least a subset of the fragments are ligated to generate shuffled nucleic acid strands. A selected strand is identified from the shuffled nucleic acid strands for a criterion. The methods of the invention can also be used to diversify proteins.
摘要翻译： 公开了一种改变核酸如RNA或DNA的方法。 该方法包括将母体核酸链断裂以产生核酸片段。 连接片段的至少一个子集以产生洗牌的核酸链。 从洗牌的核酸链中鉴定出选择的链，用于标准。 本发明的方法也可用于使蛋白多样化。
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公开(公告)号：US20100209994A1
公开(公告)日：2010-08-19
申请号：US12303047
申请日：2007-06-01
CPC分类号： C07K14/43595 , C12N9/93 , C12Y603/02003
摘要： Aggregation is a major cause of the misbehavior of proteins. A system for modifying a protein to create a more stable variant is provided. The method involves identifying non-conserved hydrophobic amino acid residues on the surface of a protein, suitable for mutating to more hydrophilic residues (e.g., charged amino acids). Any number of residues on the surface may be changed to create a variant that is more soluble, resistant to aggregation, has a greater ability to re-fold, and/or is more stable under a variety of conditions. The invention also provides GFP, streptavidin, and GST variants with an increased theoretical net charge created by the inventive technology. Kits are also provided for carrying out such modifications on any protein of interest.
摘要翻译： 聚集是蛋白质不正当行为的主要原因。 提供了用于修饰蛋白质以产生更稳定变体的系统。 该方法涉及鉴定蛋白质表面上的非保守疏水性氨基酸残基，适合于突变为更亲水残基（例如带电氨基酸）。 可以改变表面上的任何数量的残基，以产生更可溶，耐聚集，更大的再折叠能力和/或在各种条件下更稳定的变体。 本发明还提供具有由本发明技术产生的增加的理论净电荷的GFP，链霉抗生物素蛋白和GST变体。 还提供了用于对任何感兴趣的蛋白进行这些修饰的试剂盒。
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公开(公告)号：US20090203530A1
公开(公告)日：2009-08-13
申请号：US11916710
申请日：2006-06-07
CPC分类号： C12N15/1068 , C12N15/1062
摘要： The invention provides a method for producing polymers having a desirable property, for example, catalytic activity or binding activity, via evolutionary nucleic acid-mediated chemistry.
摘要翻译： 本发明提供了一种通过进化核酸介导化学制备具有所需性质，例如催化活性或结合活性的聚合物的方法。
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50.发明申请公开(公告)号：US20080318807A1
公开(公告)日：2008-12-25
申请号：US11917609
申请日：2006-06-16
CPC分类号： C12N15/1068
摘要： The present invention provides methods and compositions for performing multi-step nucleic acid mediated synthesis of a highly diverse collection of molecules, for example, small molecules and polymers. In the method, in at least two steps, multiple reaction intermediates and/or products are produced in the same step by different chemical reactions.
摘要翻译： 本发明提供用于进行多步骤核酸介导的高度多样化分子收集的方法和组合物，例如小分子和聚合物。 在该方法中，在至少两个步骤中，通过不同的化学反应在同一步骤中产生多个反应中间体和/或产物。
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