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    Discovery of candidate biomarkers of in vivo apoptosis by global profiling of caspase cleavage sites
    11.
    发明授权
    Discovery of candidate biomarkers of in vivo apoptosis by global profiling of caspase cleavage sites 有权
    通过胱天蛋白酶切割位点的全局分析发现候选生物标志物的体内凋亡

    公开(公告)号：US09493522B2

    公开(公告)日：2016-11-15

    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： G01N31/00 ,  G01N33/53 ,  C07K14/47 ,  C12Q1/37 ,  G01N33/573 ,  G01N33/574 ,  G01N33/68

    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。

    SPECIFIC N-TERMINAL LABELING OF PEPTIDES AND PROTEINS IN COMPLEX MIXTURES
    12.
    发明申请
    SPECIFIC N-TERMINAL LABELING OF PEPTIDES AND PROTEINS IN COMPLEX MIXTURES 有权
    复合混合物中肽和蛋白质的特异性N-末端标记

    公开(公告)号：US20100143912A1

    公开(公告)日：2010-06-10

    申请号：US12524557

    申请日：2008-01-24

    申请人： James A. Wells ,  Sami Mahrus

    发明人： James A. Wells ,  Sami Mahrus

    IPC分类号： C12Q1/68 ,  C12P21/06 ,  C12Q1/37

    CPC分类号： G01N33/6803 ,  C12Q1/34

    摘要： This invention provides general methods for selective labeling of proteins on their N-termini with synthetic peptides. The methods of this invention can be applied to the global proteomic profiling of complex mixtures of proteins and polypeptides.

    摘要翻译： 本发明提供了用合成肽在其N-末端上选择性标记蛋白质的一般方法。 本发明的方法可以应用于蛋白质和多肽的复合物混合物的全局蛋白质组学分析。

    Growth hormone variants
    14.
    发明授权
    Growth hormone variants 失效
    生长激素变体

    公开(公告)号：US06780613B1

    公开(公告)日：2004-08-24

    申请号：US08479884

    申请日：1995-06-07

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

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

    IPC分类号： C12N121

    CPC分类号： C07K14/575 ,  C12N15/1058 ,  Y10S530/806 ,  Y10S530/808

    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.

    摘要翻译： 本发明提供了通过鉴定影响多肽与靶物质的活性的活性结构域来系统分析多肽的结构和功能的方法。 通过用类似物与多肽的类似多肽片段取代多肽的选定氨基酸片段来确定此类活性结构域。 与亲本多肽相比，类似物与目标物质具有不同的活性。 将段取代的多肽的活性与目标的亲本多肽的相同活性进行比较。 这些活性的比较提供了活性结构域在亲本多肽中的位置的指示。 本发明还提供鉴定亲本多肽活性结构域内活性氨基酸残基的方法。 该方法包括用扫描氨基酸取代母体多肽的活性结构域内的氨基酸残基之一，并测定与靶物质形成的残基取代的多肽。 本发明还提供了包含片段取代的和残基取代的生长激素，催乳素和胎盘乳糖的多肽变体。

    Heregulin variants
    15.
    发明授权
    Heregulin variants 失效
    特殊蛋白变体

    公开(公告)号：US6136558A

    公开(公告)日：2000-10-24

    申请号：US20880

    申请日：1998-02-09

    申请人： Marcus D. Ballinger ,  Jennifer T. Jones ,  Wayne J. Fairbrother ,  Mark X. Sliwkowski ,  James A. Wells

    发明人： Marcus D. Ballinger ,  Jennifer T. Jones ,  Wayne J. Fairbrother ,  Mark X. Sliwkowski ,  James A. Wells

    IPC分类号： C07K14/575 ,  C12P21/06

    CPC分类号： C07K14/57509

    摘要： The present invention provides heregulin variants that are capable of binding an ErbB receptor. Included in the invention are variants of human heregulins, and, in particular, variants of human heregulin-.beta.1 having enhanced affinity for the ErbB-3 and ErbB-4 receptors. These variants include at least one amino acid substitution and can include further modifications. The invention also provides nucleic acid molecules encoding heregulin variants and related vectors, host cells, pharmaceutical compositions, and methods.

    摘要翻译： 本发明提供能够结合ErbB受体的调节蛋白变体。 本发明中包括本发明的变体，尤其是具有对ErbB-3和ErbB-4受体具有增强亲和力的人类胰岛素β1的变体。 这些变体包括至少一个氨基酸取代并且可以包括进一步的修饰。 本发明还提供了编码本领域变体和相关载体的核酸分子，宿主细胞，药物组合物和方法。

    Subtilisin variants capable of cleaving substrates containing dibasic residues
    19.
    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
    20.
    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。