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公开(公告)号：US5846765A
公开(公告)日：1998-12-08
申请号：US441871
申请日：1995-05-16
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。
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62.发明授权Subtilisin variants capable of cleaving substrates containing basic residues 失效能够裂解含有碱性残基的底物的枯草杆菌蛋白酶变体
公开(公告)号：US5837516A
公开(公告)日：1998-11-17
申请号：US504265
申请日：1995-07-19
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 basic residues. Combination mutants, where Asn 62 was changed to Asp, Gly 166 was changed to Asp (N62D/G166D), and optionally Tyr 104 was changed to Asp had a larger than additive shift in specificity toward substrates containing basic residues. 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- (SEQ ID NO: 35), that was selectively cleaved in the context of a fusion protein by the N62D/G166D subtilisin variant. A particularly good substrate for N62D/G166D/Y104D would be Asn-Arg-Met-Arg-Lys- (SEQ ID NO: 76). Accordingly, these variant subtilisin are useful for cleaving fusion proteins with basic substrate linkers and processing hormones or other proteins (in vitro or in vivo) that contain basic cleavage sites.
摘要翻译： 细菌丝氨酸蛋白酶枯草杆菌蛋白酶BPN'已被突变，从而能有效地选择性地切割含有碱性残基的底物。 将Asn 62改变为Asp的组合突变体，将Gly 166改变为Asp（N62D / G166D），并且任选地将Tyr 104改变为Asp，对于含有碱性残基的底物的特异性大于添加位移。 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。 该方法鉴定了通过N62D / G166D枯草杆菌蛋白酶变体在融合蛋白的上下文中选择性切割的特别良好的底物Asn-Leu-Met-Arg-Lys-（SEQ ID NO：35）。 N62D / G166D / Y104D特别好的底物是Asn-Arg-Met-Arg-Lys-（SEQ ID NO：76）。 因此，这些变体枯草杆菌蛋白酶可用于用碱性底物接头和加工激素或其它含有碱基切割位点的蛋白质（体外或体内）切割融合蛋白。
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公开(公告)号：US5741664A
公开(公告)日：1998-04-21
申请号：US460343
申请日：1995-06-01
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.
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公开(公告)号：US5688666A
公开(公告)日：1997-11-18
申请号：US182530
申请日：1994-01-14
申请人： Steven H. Bass , Ronald Greene , Henry B. Lowman , James A. Wells
发明人： Steven H. Bass , Ronald Greene , Henry B. Lowman , James A. Wells
CPC分类号： C40B40/02 , C07K14/575 , C07K14/61 , C12N15/1037 , C12N15/62 , C07K2319/00 , C07K2319/02 , C07K2319/735 , C07K2319/75 , G01N2333/61
摘要： A method for selecting novel proteins such as growth hormone variants having altered binding properties for a growth hormone receptor molecule is provided. The method comprises fusing a gene encoding a hormone to the carboxy terminal domain of the gene III coat protein of the filamentous phage M13. The gene fusion is mutated to form a library of structurally related fusion proteins that are expressed in low quantity on the surface of a phagemid particle. Biological selection and screening are employed to identify novel ligands useful as drug candidates. Disposed are preferred phagemid expression vectors and selected human growth hormone variants.
摘要翻译： 提供了一种用于选择具有改变的生长激素受体分子结合特性的生长激素变体的新蛋白质的方法。 该方法包括将编码激素的基因融合到丝状噬菌体M13的基因III外壳蛋白的羧基末端结构域上。 基因融合突变以形成在噬菌粒粒子表面上以低量表达的结构相关融合蛋白的文库。 使用生物选择和筛选来鉴定可用作候选药物的新型配体。 优选的噬菌粒表达载体和选择的人生长激素变体。
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65.发明授权
公开(公告)号：US5580723A
公开(公告)日：1996-12-03
申请号：US190723
申请日：1994-02-02
IPC分类号： G01N33/53 , A61K38/24 , A61K38/27 , A61P3/10 , A61P5/00 , A61P5/06 , C07K1/00 , C07K14/00 , C07K14/435 , C07K14/47 , C07K14/575 , C07K14/61 , C07K16/00 , C12N1/21 , C12N5/10 , C12N15/00 , C12N15/09 , C12N15/10 , C12N15/12 , C12N15/16 , C12N15/18 , C12N15/62 , C12N15/63 , C12N15/70 , C12P21/02 , C12P21/06 , C12Q1/68 , C12R1/19 , C40B40/02 , G01N33/48 , G01N33/50 , G01N33/566 , G01N33/68 , G01N33/74 , G06F19/00
CPC分类号： C40B40/02 , C07K14/575 , C07K14/61 , C12N15/1037 , C12N15/1058 , C12N15/62 , G01N33/6878 , G01N33/74 , C07K2319/00 , C07K2319/75 , G01N2333/61 , 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.
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公开(公告)号：US5506107A
公开(公告)日：1996-04-09
申请号：US122548
申请日：1993-09-29
IPC分类号： A01K67/027 , A61K38/00 , A61K38/04 , A61K39/395 , A61P5/02 , A61P5/06 , A61P43/00 , C07K14/505 , C07K14/52 , C07K14/53 , C07K14/535 , C07K14/54 , C07K14/545 , C07K14/55 , C07K14/555 , C07K14/56 , C07K14/565 , C07K14/575 , C07K14/61 , C07K14/705 , C07K16/28 , C12N1/15 , C12N1/19 , C12N1/21 , C12N5/10 , C12N15/09 , C12N15/12 , C12N15/85 , C12P21/02 , G01N33/15 , G01N33/50 , G01N33/53 , G01N33/542 , G01N33/68 , G01N33/74 , G01N33/566
CPC分类号： C12N15/8509 , C07K14/505 , C07K14/535 , C07K14/5412 , C07K14/57554 , C07K14/61 , C07K16/2869 , G01N33/74 , A01K2207/15 , A01K2217/00 , A01K2217/05 , A01K2267/03 , A61K38/00 , G01N2333/61
摘要： We have discovered that growth hormones form ternary complexes with their receptors in which site 1 on the hormone first binds to one molecule of receptor and then hormone site 2 then binds to another molecule of receptor, thereby producing a 1:2 complex. We believe this phenomenon is shared by other ligands having similar conformational structure. Assays based on this phenomenon are useful for identifying ligand agonists and antagonists. Sites 1 and 2 are structurally identified to facilitate generation of amino acid sequence variants of ternary complex-forming ligands. Novel variants of growth hormone, prolactin placental lactogen and other related ligands are provided. As a result of our studies with the ternary complex we have determined that selected antibodies to the receptor for these ligands are capable of acting as ligand agonists or antagonists. Novel growth hormones and novel uses for anti-growth hormone receptor antibodies are described.
摘要翻译： PCT No.PCT / US92 / 03743 Sec。 371日期：1993年9月29日 102（e）日期1993年9月29日PCT提交1992年5月6日PCT公布。 出版物WO92 / 21029 日期：1992年11月26日。我们发现生长激素与其受体形成三元复合物，其中激素上的位点1首先与一个受体分子结合，然后激素位点2然后结合另一个受体分子，由此产生1 ：2复杂。 我们认为这种现象由具有相似构象结构的其他配体共享。 基于此现象的分析可用于鉴定配体激动剂和拮抗剂。 在结构上鉴定了位点1和2，以便于产生三元复合物形成配体的氨基酸序列变体。 提供了生长激素，催乳素胎盘乳酸和其他相关配体的新型变体。 作为我们用三元复合物研究的结果，我们确定了这些配体的受体的选定抗体能够作为配体激动剂或拮抗剂。 描述了抗生长激素受体抗体的新型生长激素和新用途。
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公开(公告)号：US5403737A
公开(公告)日：1995-04-04
申请号：US969154
申请日：1993-02-26
申请人： Lars B. Abrahmsen , John Burnier , James A. Wells
发明人： Lars B. Abrahmsen , John Burnier , James A. Wells
IPC分类号： C12N1/21 , C07K14/61 , C12N9/22 , C12N9/54 , C12N15/09 , C12N15/57 , C12P21/02 , C12R1/07 , C12R1/125 , C12N1/20 , C12N9/52 , C12N15/00
摘要： The invention relates to serine protease variants derived from precursor serine proteases via recombinant and/or chemical methods to form protease variants having improved peptide ligase activity. The invention also includes novel ligation substrates which in combination with the serine protease variants and a second ligation substrate are capable of forming a ligation product. The invention also relates to methods for forming such ligation products and the products formed thereby.
摘要翻译： PCT No.PCT / US91 / 05480 Sec。 371日期：1993年2月26日 102（e）日期1993年2月26日PCT 1991年8月6日PCT PCT。 出版物WO92 / 02615 日本1992年2月20日。本发明涉及通过重组和/或化学方法衍生自前体丝氨酸蛋白酶的丝氨酸蛋白酶变体，以形成具有改善的肽连接酶活性的蛋白酶变体。 本发明还包括与丝氨酸蛋白酶变体组合的新型连接底物，第二连接底物能够形成连接产物。 本发明还涉及形成这种连接产品的方法和由此形成的产品。
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公开(公告)号：US5371190A
公开(公告)日：1994-12-06
申请号：US90902
申请日：1993-07-12
申请人： Paul J. Carter , James A. Wells
发明人： Paul J. Carter , James A. Wells
摘要： Novel enzyme mutants are disclosed which are derived from a precursor enzyme by replacing or modifying at least one catalytic functional group of an amino acid residue in a precursor enzyme. Such mutant enzymes have a catalytic preference for substrates which provide the replaced or modified catalytic group or its equivalent such that the substrate together with the enzyme mutant assists in its own catalysis.
摘要翻译： 公开了通过置换或修饰前体酶中氨基酸残基的至少一个催化官能团而衍生自前体酶的新型酶突变体。 这样的突变酶对提供替代或修饰的催化基团或其等同物的底物具有催化倾向，使得底物与酶突变体一起有助于其自身的催化。
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公开(公告)号：US5371008A
公开(公告)日：1994-12-06
申请号：US90472
申请日：1993-07-12
申请人： Paul J. Carter , James A. Wells
发明人： Paul J. Carter , James A. Wells
CPC分类号： C12R1/07 , C11D3/386 , C12N15/102 , C12N15/75 , C12N9/54
摘要： Novel enzyme mutants are disclosed which are derived from a precursor enzyme by replacing or modifying at least one catalytic functional group of an amino acid residue in a precursor enzyme. Such mutant enzymes have a catalytic preference for substrates which provide the replaced or modified catalytic group or its equivalent such that the substrate together with the enzyme mutant assists in its own catalysis.
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公开(公告)号：USD310570S
公开(公告)日：1990-09-11
申请号：US153650
申请日：1988-02-08
申请人： James A. Wells
设计人： James A. Wells
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