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1.发明申请Feeding buffers, systems, and methods for in vitro synthesis of biomolecules 审中-公开饲养缓冲液，系统和生物分子体外合成方法公开(公告)号：US20060110788A1
公开(公告)日：2006-05-25
申请号：US11240651
申请日：2005-10-03
申请人： Wieslaw Kudlicki , Shiranthi Keppetipola , Julia Fletcher , Ashley Getbehead , Federico Katzen , Laura Vozza-Brown
发明人： Wieslaw Kudlicki , Shiranthi Keppetipola , Julia Fletcher , Ashley Getbehead , Federico Katzen , Laura Vozza-Brown
摘要： Compositions, methods and kits for in vitro systems for synthesis of biomolecules such as polypeptides, are provided herein. Cell extracts that provide enhanced yields of soluble proteins using in vitro protein synthesis methods are provided. The invention also includes methods for producing high yields of proteins by the addition of a feeding solution that includes amino acids and an energy source to an ongoing in vitro synthesis system. The invention also includes methods of using a high-yield in vitro synthesis system to produce large quantities of proteins with incorporated labeled amino acids for analysis by methods such as by NMR. The invention further includes vectors for enhanced production of proteins from nucleic acid templates using in vitro synthesis systems.
摘要翻译： 本文提供了用于合成生物分子如多肽的体外系统的组合物，方法和试剂盒。 提供了使用体外蛋白质合成方法提供增加的可溶性蛋白质产量的细胞提取物。 本发明还包括通过添加包括氨基酸和进入体外合成系统的能量源的进料溶液来生产高产量蛋白质的方法。 本发明还包括使用高产量体外合成系统产生大量具有掺入标记氨基酸的蛋白质以通过诸如NMR的方法进行分析的方法。 本发明还包括使用体外合成系统从核酸模板增强蛋白质生产的载体。
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公开(公告)号：US20090161828A1
公开(公告)日：2009-06-25
申请号：US12333191
申请日：2008-12-11
IPC分类号： G01N23/207 , C07K14/00 , C12N9/10 , C12N9/48 , C12N9/16
摘要： Systems and methods are provided for producing a protein of interest that is typically not amenable to expression in soluble form in in vitro expression systems. In some aspects, the invention provides methods of synthesizing proteins using in vitro protein synthesis systems that include a scaffold protein such as apolipoprotein or an amphipathic alpha helix containing (“AAHC”) protein, in which higher yields of soluble protein are produced than in the absence of the scaffold protein. The scaffold proteins may be provided in an in vitro protein synthesis system associated with lipid or not associated with lipid. The scaffold protein may be provided as a protein per se or may be encoded by a nucleic acid template and co-expressed with the protein of interest. The invention also provides compositions and kits for synthesis of proteins in soluble form, in which the compositions and kits include cell extracts for protein expression and isolation.
摘要翻译： 提供的系统和方法用于产生通常不适于在体外表达系统中以可溶形式表达的目的蛋白质。 在一些方面，本发明提供了使用体外蛋白质合成系统合成蛋白质的方法，所述体外蛋白质合成系统包括诸如载脂蛋白或含有（“AAHC”）蛋白质的两亲性α螺旋的支架蛋白，其中产生比在 缺少支架蛋白。 支架蛋白质可以提供在与脂质相关的体外蛋白质合成系统中或与脂质不相关。 支架蛋白质可以作为蛋白质本身提供，或者可以由核酸模板编码并与感兴趣的蛋白质共表达。 本发明还提供用于合成可溶形式的蛋白质的组合物和试剂盒，其中组合物和试剂盒包括用于蛋白质表达和分离的细胞提取物。
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公开(公告)号：US20100233782A1
公开(公告)日：2010-09-16
申请号：US12576880
申请日：2009-10-09
摘要： Systems and methods are provided for producing a protein of interest that is typically not amenable to expression in soluble form in in vitro expression systems. In some aspects, the invention provides methods of synthesizing proteins using in vitro protein synthesis systems that include a scaffold protein such as apolipoprotein or an amphipathic alpha helix containing (“AAHC”) protein, in which higher yields of soluble protein are produced than in the absence of the scaffold protein. The scaffold proteins may be provided in an in vitro protein synthesis system associated with lipid or not associated with lipid. The scaffold protein may be provided as a protein per se or may be encoded by a nucleic acid template and co-expressed with the protein of interest. The invention also provides compositions and kits for synthesis of proteins in soluble form, in which the compositions and kits include cell extracts for protein expression and isolation.
摘要翻译： 提供的系统和方法用于产生通常不适于在体外表达系统中以可溶形式表达的目的蛋白质。 在一些方面，本发明提供了使用体外蛋白质合成系统合成蛋白质的方法，所述体外蛋白质合成系统包括诸如载脂蛋白或含有（“AAHC”）蛋白质的两亲性α螺旋的支架蛋白，其中产生比在 缺少支架蛋白。 支架蛋白质可以提供在与脂质相关的体外蛋白质合成系统中或与脂质不相关。 支架蛋白质可以作为蛋白质本身提供，或者可以由核酸模板编码并与感兴趣的蛋白质共表达。 本发明还提供用于合成可溶形式的蛋白质的组合物和试剂盒，其中组合物和试剂盒包括用于蛋白质表达和分离的细胞提取物。
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公开(公告)号：US20060211083A1
公开(公告)日：2006-09-21
申请号：US11336644
申请日：2006-01-20
CPC分类号： C12P19/34 , C12P21/005
摘要： Provided herein are products and processes for efficiently synthesizing biomolecules in vitro using cell-free extracts derived from mammalian cells and insect cells. In an embodiment, provided is a process for preparing a cell-free extract from insect cells and mammalian cells that efficiently synthesizes post-translationally modified target proteins (e.g., glycosylated target proteins). In some embodiments, a ribonucleic acid is synthesized in vitro that comprises a cap, a 5′ untranslated region comprising an 18S rRNA binding ribonucleotide sequence, and a target ribonucleotide sequence. It has been determined that such ribonucleic acids result in efficient in vitro synthesis of a target protein using cell-free extracts derived from non-rabbit mammalian cells and insect cells.
摘要翻译： 本文提供了使用来自哺乳动物细胞和昆虫细胞的无细胞提取物在体外有效合成生物分子的产物和方法。 在一个实施方案中，提供了从昆虫细胞和哺乳动物细胞制备无细胞提取物的方法，其有效合成翻译后修饰的靶蛋白（例如糖基化靶蛋白）。 在一些实施方案中，在体外合成核糖核酸，其包含帽，包含18S rRNA结合核糖核苷酸序列的5'非翻译区和靶核糖核苷酸序列。 已经确定，使用来自非兔哺乳动物细胞和昆虫细胞的无细胞提取物，这种核糖核酸导致靶蛋白的有效体外合成。
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公开(公告)号：US20060084136A1
公开(公告)日：2006-04-20
申请号：US11181023
申请日：2005-07-14
IPC分类号： C12P21/06
CPC分类号： C12P21/02
摘要： The present invention relates to in vitro protein synthesis (IVPS) systems, particularly such systems using suppressor tRNAs and rare codon tRNAs to extend translation of an open reading frame into fusion protein elements, thereby generating fusion proteins in vitro. The invention also provides methods, compositions and kits using the IVPS systems of the invention, and proteins produced using the methods, compositions, kits and IVPS systems of the invention.
摘要翻译： 本发明涉及体外蛋白质合成（IVPS）系统，特别是使用抑制子tRNA和稀有密码子tRNA的系统将开放阅读框的翻译扩展到融合蛋白质元件中，从而在体外产生融合蛋白。 本发明还提供使用本发明的IVPS系统的方法，组合物和试剂盒，以及使用本发明的方法，组合物，试剂盒和IVPS系统产生的蛋白质。
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公开(公告)号：US20080248565A1
公开(公告)日：2008-10-09
申请号：US12040798
申请日：2008-02-29
摘要： Systems and methods are provided for producing a protein of interest that is typically not amenable to expression in soluble form in in vitro expression systems. In some aspects, the invention provides methods of synthesizing proteins using in vitro protein synthesis systems that include a scaffold protein such as apolipoprotein or an amphipathic alpha helix containing (“AAHC”) protein, in which higher yields of soluble protein are produced than in the absence of the scaffold protein. The scaffold proteins may be provided in an in vitro protein synthesis system associated with lipid or not associated with lipid. The scaffold protein may be provided as a protein per se or may be encoded by a nucleic acid template and co-expressed with the protein of interest. The invention also provides compositions and kits for synthesis of proteins in soluble form, in which the compositions and kits include cell extracts for protein expression and isolation.
摘要翻译： 提供的系统和方法用于产生通常不适于在体外表达系统中以可溶形式表达的目的蛋白质。 在一些方面，本发明提供了使用体外蛋白质合成系统合成蛋白质的方法，所述体外蛋白质合成系统包括诸如载脂蛋白或含有（“AAHC”）蛋白质的两亲性α螺旋的支架蛋白，其中产生比在 缺少支架蛋白。 支架蛋白质可以提供在与脂质相关的体外蛋白质合成系统中或与脂质不相关。 支架蛋白质可以作为蛋白质本身提供，或者可以由核酸模板编码并与感兴趣的蛋白质共表达。 本发明还提供用于合成可溶形式的蛋白质的组合物和试剂盒，其中组合物和试剂盒包括用于蛋白质表达和分离的细胞提取物。
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7.发明申请IN VITRO PROTEIN SYNTHESIS SYSTEMS FOR MEMBRANE PROTEINS THAT INCLUDE ADOLIPOPROTEINS AND PHOSPHOLIPID-ADOLIPOPROTEIN PARTICLES 审中-公开用于含有ADOLIPOPROTEINS和磷脂 - ADOLIPOPROTEIN颗粒的膜蛋白的体外蛋白合成系统公开(公告)号：US20070117179A1
公开(公告)日：2007-05-24
申请号：US11535960
申请日：2006-09-27
IPC分类号： C12P21/06
CPC分类号： C12P21/00
摘要： In vitro protein synthesis systems and methods are provided that produce membrane proteins in soluble form. In some aspects, the invention provides methods of synthesizing proteins using in vitro protein synthesis systems that include an apolipoprotein, in which higher yields of soluble protein are produced than in the absence of the apolipoprotein. Apolipoproteins useful in the present invention include naturally occurring apolipoproteins, as well as sequence variants of wild-type apolipoproteins, and engineered apolipoproteins. The apolipoproteins can be provided in an in vitro protein synthesis system associated with lipid or not associated with lipid. The invention also provides compositions and kits for synthesis of proteins in soluble form, in which the compositions and kits include cell extracts for protein translation and at least one apolipoprotein biomolecule.
摘要翻译： 提供体外蛋白质合成系统和方法，其产生可溶形式的膜蛋白质。 在一些方面，本发明提供使用体外蛋白质合成系统合成蛋白质的方法，所述体外蛋白质合成系统包括与不存在载脂蛋白时产生更高产率的可溶性蛋白质的载脂蛋白。 可用于本发明的载脂蛋白包括天然存在的载脂蛋白，以及野生型载脂蛋白和工程化载脂蛋白的序列变体。 载脂蛋白可以在与脂质相关的体外蛋白质合成系统中提供，或与脂质不相关。 本发明还提供用于合成可溶形式的蛋白质的组合物和试剂盒，其中组合物和试剂盒包括用于蛋白质翻译的细胞提取物和至少一种载脂蛋白生物分子。
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8.发明申请Compositions and methods for synthesizing, purifying, and detecting biomolecules 审中-公开用于合成，纯化和检测生物分子的组合物和方法公开(公告)号：US20050136449A1
公开(公告)日：2005-06-23
申请号：US10954951
申请日：2004-10-01
IPC分类号： C12N20060101 , C12N9/52 , C12N9/90 , C12Q1/68 , G01N33/554 , G01N33/569
CPC分类号： C12N9/90
摘要： Compositions and methods that can reduce or eliminate purification and detection difficulties related to SlyD polypeptides are disclosed. The compositions include cells that lack or contain a reduced amount of SlyD, cells that contain SlyD mutated to reduce or eliminate its ability to bind biarsenical reagents; anti-SlyD antibodies, and kits containing the same. The disclosed compositions can be used, e.g., to purify and detect recombinant polypeptides having polyhistidine or polycysteine tags.
摘要翻译： 公开了可降低或消除与SlyD多肽相关的纯化和检测困难的组合物和方法。 组合物包括缺乏或含有减少量的SlyD的细胞，含有突变的SlyD的细胞以减少或消除其结合双相试剂的能力; 抗SlyD抗体，以及含有该抗体的试剂盒。 所公开的组合物可用于例如纯化和检测具有多组氨酸或多聚半胱氨酸标签的重组多肽。
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公开(公告)号：US06448033B1
公开(公告)日：2002-09-10
申请号：US08590729
申请日：1996-01-24
申请人： Wieslaw Kudlicki , Gisela Kramer , Boyd Hardesty
发明人： Wieslaw Kudlicki , Gisela Kramer , Boyd Hardesty
IPC分类号： C12P2106
CPC分类号： C07K1/1077 , C12N9/10 , C12P21/02 , G01N33/533
摘要： The present invention provides a method of synthesizing a fluorescently labeled protein in a cell free protein synthesis system, comprising the steps of: (a) incubating a sample of ribosomes obtained from a cell-free extract with plasmid DNA containing a coding sequence for a protein of interest, said sample incubated in a coupled transcription/translation medium together with a aminoacyl tRNA having fluorescent label; (b) partially purifying said fluorescently labeled protein by separating newly synthesized, fluorescently-labeled protein from other fluorescent components within said sample; (c) measuring the amount of protein synthesized; (d) determining fluorescence of newly synthesized protein; and (e) determining the biological activity of the newly synthesized protein.
摘要翻译： 本发明提供了一种在无细胞蛋白质合成系统中合成荧光标记的蛋白质的方法，包括以下步骤：（a）将从无细胞提取物获得的核糖体样品与含有蛋白质编码序列的质粒DNA 所述样品与偶联的转录/翻译培养基一起孵育，同时具有荧光标记的氨酰基tRNA; （b）通过将新合成的荧光标记的蛋白质与所述样品中的其他荧光成分分离，部分纯化所述荧光标记的蛋白质; （c）测量合成的蛋白质的量; （d）确定新合成的蛋白质的荧光; 和（e）确定新合成的蛋白质的生物活性。
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