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71 条记录 (耗时 0.043 秒)

    Subtilisin variants capable of cleaving substrates containing dibasic residues
    23.
    发明授权
    Subtilisin variants capable of cleaving substrates containing dibasic residues 失效
    能够切割含有二碱基残基的底物的枯草杆菌蛋白酶变体

    公开(公告)号：US5780285A

    公开(公告)日：1998-07-14

    申请号：US398028

    申请日：1995-03-03

    申请人： Marcus D. Ballinger ,  James A. Wells

    发明人： Marcus D. Ballinger ,  James A. Wells

    IPC分类号： C12N9/54 ,  C12N9/64 ,  C12N15/57 ,  C12N15/62 ,  C12P21/06 ,  C12N9/56 ,  C12N15/75

    CPC分类号： C12N9/6454 ,  C12N15/62 ,  C12N9/54 ,  C12P21/06 ,  C07K2319/00 ,  C07K2319/50 ,  C07K2319/75

    摘要： The bacterial serine protease, subtilisin BPN', has been mutated so that it will efficiently and selectively cleave substrates containing dibasic residues. A combination mutant, where Asn 62 was changed to Asp and Gly 166 was changed to Asp (N62D/G166D), had a larger than additive shift in specificity toward dibasic substrates. Suitable substrates of the variant subtilisin were revealed by sorting a library of phage particles (substrate phage) containing five contiguous randomized residues. This method identified a particularly good substrate, Asn-Leu-Met-Arg-Lys-, that was selectively cleaved in the context of a fusion protein by the N62D/G166D subtilisin variant. Accordingly, this variant subtilisin may be useful for cleaving fusion proteins with dibasic substrate linkers and processing hormones or other proteins (in vitro or in vivo) that contain dibasic cleavage sites.

    摘要翻译： 细菌丝氨酸蛋白酶枯草杆菌蛋白酶BPN'已经被突变，使得其将有效地和选择性地裂解含有二元残基的底物。 将Asn 62改变为Asp和Gly 166的组合突变体改变为Asp（N62D / G166D），其特异性对于二元底物的添加偏移大。 通过分选含有五个连续随机残留物的噬菌体颗粒（底物噬菌体）文库来揭示变体枯草杆菌蛋白酶的合适底物。 该方法鉴定了特异性良好的底物Asn-Leu-Met-Arg-Lys-，其被N62D / G166D枯草杆菌蛋白酶变体在融合蛋白的上下文中选择性地切割。 因此，这种变体枯草杆菌蛋白酶可用于用含有二碱基切割位点的二碱基底物接头和加工激素或其他蛋白质（体外或体内）切割融合蛋白。

    Method of selection of proteolytic cleavage sites by directed evolution and phagemid display
    24.
    发明授权
    Method of selection of proteolytic cleavage sites by directed evolution and phagemid display 失效
    通过定向进化和噬菌粒展示来选择蛋白水解切割位点的方法

    公开(公告)号：US5780279A

    公开(公告)日：1998-07-14

    申请号：US418928

    申请日：1995-04-05

    申请人： David J. Matthews ,  James A. Wells ,  Mark J. Zoller

    发明人： David J. Matthews ,  James A. Wells ,  Mark J. Zoller

    IPC分类号： C12N15/10 ,  C40B40/02 ,  C07K14/435 ,  C12N15/12

    CPC分类号： C40B40/02 ,  C12N15/1037 ,  C07K2319/00 ,  C07K2319/02 ,  C07K2319/735 ,  C07K2319/75

    摘要： A method for identifying and selecting novel substrates for enzymes is provided. The method comprises constructing a gene fusion comprising DNA encoding a polypeptide fused to DNA encoding a substrate peptide, which in turn is fused to DNA encoding at least a portion of a phage coat protein. The DNA encoding the substrate peptide is mutated at one or more codons thereby generating a family of mutants. The fusion protein is expressed on the surface of a phagemid particle and subjected to chemical or enzymatic modification of the substrate peptide. Those phagemid particles which have been modified are then separated from those that have not.

    摘要翻译： 提供了用于鉴定和选择酶的新基质的方法。 该方法包括构建包含编码与编码底物肽的DNA融合的多肽的DNA的基因融合物，其又与编码至少一部分噬菌体外壳蛋白质的DNA融合。 编码底物肽的DNA以一个或多个密码子突变，从而产生突变体家族。 融合蛋白在噬菌粒的表面上表达并进行底物肽的化学或酶修饰。 然后将已经被修饰的那些噬菌粒颗粒与那些未被修饰的噬菌粒分离出来。

    Method for identifying active domains and amino acid residues in polypeptides and hormone variants
    25.
    发明授权
    Method for identifying active domains and amino acid residues in polypeptides and hormone variants 失效

    公开(公告)号：US5766854A

    公开(公告)日：1998-06-16

    申请号：US483039

    申请日：1995-06-06

    申请人： James A. Wells ,  Brian C. Cunningham

    发明人： James A. Wells ,  Brian C. Cunningham

    摘要： The invention provides methods for the systematic analysis of the structure and function of polypeptides by identifying active domains which influence the activity of the polypeptide with a target substance. Such active domains are determined by substituting selected amino acid segments of the polypeptide with an analogous polypeptide segment from an analog to the polypeptide. The analog has a different activity with the target substance as compared to the parent polypeptide. The activities of the segment-substituted polypeptides are compared to the same activity for the parent polypeptide for the target. A comparison of such activities provides an indication of the location of the active domain in the parent polypeptide. The invention also provides methods for identifying the active amino acid residues within the active domain of the parent polypeptide. The method comprises substituting a scanning amino acid for one of the amino acid residues within the active domain of the parent polypeptide and assaying the residue-substituted polypeptide so formed with a target substance. The invention further provides polypeptide variants comprising segment-substituted and residue-substituted growth hormones, prolactins and placental lactogens.

    DISCOVERY OF CANDIDATE BIOMARKERS OF IN VIVO APOPTOSIS BY GLOBAL PROFILING OF CASPASE CLEAVAGE SITES
    30.
    发明申请
    DISCOVERY OF CANDIDATE BIOMARKERS OF IN VIVO APOPTOSIS BY GLOBAL PROFILING OF CASPASE CLEAVAGE SITES 有权
    通过CASPASE CLEAVAGE SITES的全球分析发现病毒性生物标志物

    公开(公告)号：US20120028266A1

    公开(公告)日：2012-02-02

    申请号：US13016710

    申请日：2011-01-28

    申请人： James A. Wells ,  Sami Mahrus

    发明人： James A. Wells ,  Sami Mahrus

    IPC分类号： G01N33/566 ,  C07K7/06 ,  C12Q1/00 ,  C07K16/18 ,  G01N33/577 ,  C12Q1/37 ,  C07K14/47 ,  C07K7/08

    CPC分类号： C07K14/47 ,  C07K14/4747 ,  C12Q1/37 ,  G01N33/573 ,  G01N33/574 ,  G01N33/6848 ,  G01N2333/96466 ,  G01N2510/00

    摘要： The present invention relates to the discovery of novel biomarkers of in vivo apoptosis based on a large number of caspase-like cleavage sites. These biomarkers are useful for detection and quantification of apoptosis in a biological sample. The invention also provides synthetic peptides and proteins corresponding to neo-epitopes created by proteolytic processing of these cleavage sites. The synthetic peptides can be used as standards to enable identification and quantitation of these biomarkers using mass spectrometry. The synthetic proteins can be used to generate antibodies and other binding reagents specific for these biomarkers. Methods for detecting apoptosis as well as for diagnosing or for providing a prognosis for a disease or disease state characterized by apoptosis are also provided herein. Finally, the invention provides compositions and kits for performing the methods of the invention.

    摘要翻译： 本发明涉及基于大量胱天蛋白酶样切割位点发现体内细胞凋亡的新生物标志物。 这些生物标志物可用于检测和定量生物样品中的细胞凋亡。 本发明还提供了对应于通过这些切割位点的蛋白水解加工产生的新表位的合成肽和蛋白质。 合成肽可以用作标准物，以便能够使用质谱鉴定和定量这些生物标志物。 合成蛋白质可用于产生对这些生物标志物特异性的抗体和其他结合试剂。 本文还提供了检测细胞凋亡以及用于诊断或提供特征为凋亡的疾病或疾病状态的预后的方法。 最后，本发明提供了用于实施本发明方法的组合物和试剂盒。