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1. 发明申请

US20090280560A1 Yeast cell surface display of proteins and uses thereof 有权
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US20090280560A1
公开(公告)日：2009-11-12
申请号：US12316916
申请日：2008-12-16
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C12N15/63 , C07K9/00 , C07K16/00 , C07K14/725
CPC分类号： C07K14/705 , C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。每个酵母细胞的外壁可以显示大约104个蛋白质凝集素。天然凝集素用作特异性粘附接触，以在交配期间融合相反交配型的酵母细胞。实际上，酵母已经发展出蛋白质 - 蛋白质结合的平台，而没有来自细胞壁组分的空间位阻。作为一个实施方案，将scFv抗体片段连接到Aga2p凝集素上有效地模拟了免疫系统中B细胞的抗体的细胞表面显示，用于体内的亲和力成熟。作为另一个实施方案，可以通过这种方法分离T细胞受体突变体，其有效地显示在酵母细胞表面上，提供通过文库筛选来改变T细胞受体结合亲和力和特异性的方法。

2. 发明授权

US07465787B2 Yeast cell surface display of proteins and uses thereof 有权
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US07465787B2
公开(公告)日：2008-12-16
申请号：US10738454
申请日：2003-12-16
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C07K14/37 , C07K14/39 , C07K16/28 , C07K16/46 , C07K17/00 , C07K17/02 , C07K17/06 , C12Q1/02 , C12Q1/68 , C12N1/19 , C12N15/63 , C12N15/64
CPC分类号： C07K14/705 , C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 10 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。每个酵母细胞的外壁可以显示约10个蛋白质凝集素。天然凝集素用作特异性粘附接触，以在交配期间融合相反交配型的酵母细胞。实际上，酵母已经发展出蛋白质 - 蛋白质结合的平台，而没有来自细胞壁组件的空间位阻。

3. 发明授权

US06759243B2 High affinity TCR proteins and methods 有权
标题翻译：高亲和力TCR蛋白和方法
公开(公告)号：US06759243B2
公开(公告)日：2004-07-06
申请号：US09731242
申请日：2000-12-06
申请人： David M. Kranz , K. Dane Wittrup , Phillip D. Holler
发明人： David M. Kranz , K. Dane Wittrup , Phillip D. Holler
IPC分类号： C12N510
CPC分类号： C40B40/02 , C07K14/7051 , C12N15/1037 , G01N33/68 , G01N33/6842 , G01N33/6854 , G01N2333/39
摘要： T cell receptors (TCRs) that have higher affinity for a ligand than wild type TCRs are provided. These high affinity TCRs are formed by mutagenizing a T cell receptor protein coding sequence to generate a variegated population of mutants of the T cell receptor protein coding sequence; transforming the T cell receptor mutant coding sequence into yeast cells; inducing expression of the T cell receptor mutant coding sequence on the surface of yeast cells; and selecting those cells expressing T cell receptor mutants that have higher affinity for the peptide/MHC ligand than the wild type T cell receptor protein. The high affinity TCRs can be used in place of an antibody or single chain antibody.
摘要翻译：提供了比野生型TCR对配体具有更高亲和力的T细胞受体（TCR）。通过诱变T细胞受体蛋白质编码序列产生T细胞受体蛋白编码序列突变体的杂色群体，形成这些高亲和性TCR。将T细胞受体突变体编码序列转化为酵母细胞; 在酵母细胞表面诱导T细胞受体突变体编码序列的表达; 并选择那些表达与野生型T细胞受体蛋白相比对肽/ MHC配体具有更高亲和力的T细胞受体突变体的细胞。高亲和性TCR可用于代替抗体或单链抗体。

4. 发明授权

US06699658B1 Yeast cell surface display of proteins and uses thereof 失效
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US06699658B1
公开(公告)日：2004-03-02
申请号：US09009388
申请日：1998-01-20
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C12Q168
CPC分类号： C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance, from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。每个酵母细胞的外壁可显示约10 4个蛋白质凝集素。天然凝集素用作特异性粘附接触，以在交配期间融合相反交配型的酵母细胞。实际上，酵母已经发展出蛋白质 - 蛋白质结合的平台，没有空间位阻，来自细胞壁组分。作为一个实施方案，将scFv抗体片段连接到Aga2p凝集素上有效地模拟了免疫系统中B细胞的抗体的细胞表面显示，用于体内的亲和力成熟。作为另一个实施方案，可以通过这种方法分离T细胞受体突变体，其有效地显示在酵母细胞表面上，提供通过文库筛选来改变T细胞受体结合亲和力和特异性的方法。

5. 发明授权

US06577405B2 Phase profilometry system with telecentric projector 失效
标题翻译：具有远心投影仪的相轮廓测量系统
公开(公告)号：US06577405B2
公开(公告)日：2003-06-10
申请号：US09754991
申请日：2001-01-05
申请人： David M. Kranz , Eric P. Rudd , David Fishbaine , Carl E. Haugan
发明人： David M. Kranz , Eric P. Rudd , David Fishbaine , Carl E. Haugan
IPC分类号： G01B1124
CPC分类号： H04N5/372 , G01B11/2518 , G01B11/30
摘要： An optical system for computing a height of a target on a surface includes a light projector for projecting light. The light passes through a patterned reticle and a projector lens so as to illuminate the target with an image of the pattern. The light is projected telecentrically between the reticle and the projector lens, and a camera is positioned along a receive optical path. The camera receives an image of the target through a receive lens. The target and the pattern move at least three times with respect to each other, and the camera acquires an image of the object at each of at least three positions.
摘要翻译：用于计算表面上的目标的高度的光学系统包括用于投射光的投光器。光通过图案化的掩模版和投影仪透镜，以用图案的图像照亮目标。光线以中心方式投影在标线片和投影仪透镜之间，并且相机沿着接收光路定位。相机通过接收镜头接收目标的图像。目标和图案相对于彼此移动至少三次，并且相机在至少三个位置中的每一个处获取对象的图像。

6. 发明授权

US06331391B1 Yeast cell surface display of proteins and uses thereof 失效
公开(公告)号：US06331391B1
公开(公告)日：2001-12-18
申请号：US09009388
申请日：1998-01-20
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C12Q168
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.

7. 发明授权

US06300065B1 Yeast cell surface display of proteins and uses thereof 有权
标题翻译：蛋白质的酵母细胞表面展示及其用途
公开(公告)号：US06300065B1
公开(公告)日：2001-10-09
申请号：US09140084
申请日：1998-08-26
申请人： Michele C. Kieke , K. Dane Wittrup , Eric T. Boder , David M. Kranz , Eric Shusta
发明人： Michele C. Kieke , K. Dane Wittrup , Eric T. Boder , David M. Kranz , Eric Shusta
IPC分类号： C12P2102
CPC分类号： C40B40/02 , C12N15/1037 , G01N33/68 , G01N33/6845 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. Attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening. As another embodiment, the selection method identifies proteins displayed at higher levels as proteins that are secreted at higher efficiency and that are more stable.
摘要翻译：本发明提供了一种遗传方法，用于以能够与大分子结合的形式将多肽连接到酵母细胞壁。将该方法与荧光激活细胞分选相结合提供了选择具有增加或降低的对另一分子的亲和力，改变的特异性或条件结合的蛋白质的方法。还提供了目的多肽的N末端与酵母Aga2p细胞壁蛋白的C末端的遗传融合的方法。将scFv抗体片段连接到Aga2p凝集素上有效地模拟了免疫系统中B细胞的抗体的细胞表面显示，用于体内亲和力成熟。 T细胞受体突变体可通过这种有效显示在酵母细胞表面上的方法分离，提供通过文库筛选来改变T细胞受体结合亲和力和特异性的方法。作为另一个实施方案，选择方法鉴定在较高水平上显示的蛋白质作为更高效率分泌并且更稳定的蛋白质。

8. 发明授权

US5580961A Heterodimeric T lymphocyte receptor subunit 失效
标题翻译：异二聚体T淋巴细胞受体亚单位
公开(公告)号：US5580961A
公开(公告)日：1996-12-03
申请号：US210326
申请日：1994-03-18
申请人： Haruo Saito , David M. Kranz , Herman N. Eisen , Susumu Tonegawa
发明人： Haruo Saito , David M. Kranz , Herman N. Eisen , Susumu Tonegawa
IPC分类号： C07K14/725 , C07K14/00
CPC分类号： C07K14/7051
摘要： Disclosed is a heterodimeric T lymphocyte receptor subunit. The subunit consists of variable, joining, constant, transmembrane, and cytoplasmic regions.The structure, amino acid, and nucleotide sequence of the lymphocyte receptor submit were determined using cDNA clones derived from a functional murine cytotoxic T lymphocyte clone. The genes corresponding to these cDNA are expressed and rearranged specifically in T cells and have significant sequence homologies to immunoglobulin V and C genes.T cell receptor subunits may be produced from the cDNA clones. The protein molecules may be further used for the production of T-cell clone specific antibodies.
摘要翻译：公开了异二聚体T淋巴细胞受体亚基。亚基由可变，连接，恒定，跨膜和胞质区组成。使用衍生自功能性鼠细胞毒性T淋巴细胞克隆的cDNA克隆测定淋巴细胞受体递交的结构，氨基酸和核苷酸序列。与这些cDNA相对应的基因在T细胞中特异性表达和重排，并且与免疫球蛋白V和C基因具有显着的序列同源性。 T细胞受体亚基可以从cDNA克隆产生。蛋白质分子可以进一步用于生产T细胞克隆特异性抗体。

9. 发明授权

US4970296A Heterodimeric T lymphocyte receptor 失效
标题翻译：异二聚体T淋巴细胞受体
公开(公告)号：US4970296A
公开(公告)日：1990-11-13
申请号：US385897
申请日：1989-07-27
申请人： Haruo Saito , David M. Kranz , Herman N. Eisen , Susumu Tonegawa
发明人： Haruo Saito , David M. Kranz , Herman N. Eisen , Susumu Tonegawa
IPC分类号： A61K38/00 , A61K39/00 , C07K14/725
CPC分类号： C07K14/7051 , A61K38/00 , A61K39/00
摘要： Disclosed is a heterodimeric T lymphocyte receptor comprising an alpha and a beta subunit. Each subunit consists of a signal peptide, variable, joining, constant, transmembrane, and cytoplasmic regions. The two subunits are connected by a disulfide bond between cysteine residues located between the constant and transmembrane region.The structure, amino acid, and nucleotide sequence of the lymphocyte receptor were determined using CDNA clones derived from a functional murine cytotoxic T lymphocyte clone. The genes corresponding to these cDNA are expressed and rearranged specifically in T cells and have significant sequence homologies to immunoglobulin V and C genes.Both the T cell receptor protein and its subunits may be produced from the cDNA clones. The protein molecules may be further used for the production of T-cell clone specific antibodies.
摘要翻译：公开了包含α和β亚基的异二聚体T淋巴细胞受体。每个亚基由信号肽，可变，连接，恒定，跨膜和细胞质区域组成。两个亚基通过位于恒定区和跨膜区之间的半胱氨酸残基之间的二硫键连接。使用衍生自功能性鼠细胞毒性T淋巴细胞克隆的CDNA克隆测定淋巴细胞受体的结构，氨基酸和核苷酸序列。与这些cDNA相对应的基因在T细胞中特异性表达和重排，并且与免疫球蛋白V和C基因具有显着的序列同源性。 T细胞受体蛋白及其亚基都可以从cDNA克隆产生。蛋白质分子可以进一步用于生产T细胞克隆特异性抗体。

10. 发明授权

US08372636B2 Yeast cell surface display of proteins and uses thereof 有权
公开(公告)号：US08372636B2
公开(公告)日：2013-02-12
申请号：US12316916
申请日：2008-12-16
申请人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
发明人： K. Dane Wittrup , David M. Kranz , Michele Kieke , Eric T. Boder
IPC分类号： C12N15/00
CPC分类号： C07K14/705 , C12N15/1037 , C40B40/02 , G01N33/68 , G01N33/6854 , G01N2333/39
摘要： The present invention provides a genetic method for tethering polypeptides to the yeast cell wall in a form accessible for binding to macromolecules. Combining this method with fluorescence-activated cell sorting provides a means of selecting proteins with increased or decreased affinity for another molecule, altered specificity, or conditional binding. Also provided is a method for genetic fusion of the N terminus of a polypeptide of interest to the C-terminus of the yeast Aga2p cell wall protein. The outer wall of each yeast cell can display approximately 104 protein agglutinins. The native agglutinins serve as specific adhesion contacts to fuse yeast cells of opposite mating type during mating. In effect, yeast has evolved a platform for protein-protein binding without steric hindrance from cell wall components. As one embodiment, attaching an scFv antibody fragment to the Aga2p agglutinin effectively mimics the cell surface display of antibodies by B cells in the immune system for affinity maturation in vivo. As another embodiment, T cell receptor mutants can be isolated by this method that are efficiently displayed on the yeast cell surface, providing a means of altering T cell receptor binding affinity and specificity by library screening.

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