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1. 发明申请

US20080112946A1 Catalytically active recombinant memapsin and methods of use thereof 失效
标题翻译：催化活性重组膜突触蛋白及其使用方法
公开(公告)号：US20080112946A1
公开(公告)日：2008-05-15
申请号：US11888920
申请日：2007-08-03
申请人： Gerald Koelsch , Jordan J. N. Tang , Lin Hong , Arun K. Ghosh , Xinli Lin
发明人： Gerald Koelsch , Jordan J. N. Tang , Lin Hong , Arun K. Ghosh , Xinli Lin
IPC分类号： A61K38/46 , C12N9/52 , C12P21/04
CPC分类号： C07K5/1021 , A61K38/00 , A61K39/00 , C07K1/1136 , C07K5/06026 , C07K5/06043 , C07K5/0806 , C07K2299/00 , C12N9/6421 , C12N9/6478 , Y02A90/26 , Y10S514/879
摘要： Methods for the production of purified, catalytically active, recombinant memapsin 2 have been developed. The substrate and subsite specificity of the catalytically active enzyme have been determined. The substrate and subsite specificity information was used to design substrate analogs of the natural memapsin 2 substrate that can inhibit the function of memapsin 2. The substrate analogs are based on peptide sequences, shown to be related to the natural peptide substrates for memapsin 2. The substrate analogs contain at least one analog of an amide bond which is not capable of being cleaved by memapsin 2. Processes for the synthesis of two substrate analogues including isosteres at the sites of the critical amino acid residues were developed and the substrate analogues, OMR99-1 and OM99-2, were synthesized. OM99-2 is based on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with the Leu-Ala peptide bond substituted by a transition-state isostere hydroxyethylene group (FIG. 1). The inhibition constant of OM99-2 is 1.6×10−9 M against recombinant pro-memapsin 2. Crystallography of memapsin 2 bond to this inhibitor was used to determine the three dimensional structure of the protein, as well as the importance of the various residues in binding. This information can be used by those skilled in the art to design new inhibitors, using commercially available software programs and techniques familiar to those in organic chemistry and enzymology, to design new inhibitors to memapsin 2, useful in diagnostics and for the treatment and/or prevention of Alzheimer's disease.
摘要翻译：已经开发了用于生产纯化的，催化活性的重组突变蛋白2的方法。已经确定了催化活性酶的底物和亚位点特异性。底物和亚位点特异性信息用于设计可以抑制膜蛋白2功能的天然memapsin 2底物的底物类似物。底物类似物基于肽序列，显示与memapsin 2的天然肽底物相关。底物类似物含有至少一个酰胺键的类似物，该类似物不能被膜蛋白2切割。开发了两个底物类似物合成的关键氨基酸残基位点处的等位基因，底物类似物OMR99- 1和OM99-2。 OM99-2基于由过渡态等电位羟基亚乙基取代的Leu-Ala肽键的八肽Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe（SEQ ID NO：28）（图1 ）。 OM99-2的抑制常数相对于重组前体蛋白2是1.6×10 -9 M。使用与该抑制剂的膜蛋白2键的结晶学来确定蛋白质的三维结构，以及各种残留物在结合中的重要性。本领域技术人员可以使用本信息来设计新的抑制剂，使用商业上可获得的有机化学和酶学方面熟悉的软件程序和技术来设计新的抑制剂2，可用于诊断和治疗和/或预防阿尔茨海默病。

2. 发明授权

US07829669B2 Catalytically active recombinant memapsin and methods of use thereof 失效
标题翻译：催化活性重组膜突触蛋白及其使用方法
公开(公告)号：US07829669B2
公开(公告)日：2010-11-09
申请号：US11888920
申请日：2007-08-03
申请人： Gerald Koelsch , Jordan J. N. Tang , Lin Hong , Arun K. Ghosh , Xinli Lin
发明人： Gerald Koelsch , Jordan J. N. Tang , Lin Hong , Arun K. Ghosh , Xinli Lin
IPC分类号： C07K1/00
CPC分类号： C07K5/1021 , A61K38/00 , A61K39/00 , C07K1/1136 , C07K5/06026 , C07K5/06043 , C07K5/0806 , C07K2299/00 , C12N9/6421 , C12N9/6478 , Y02A90/26 , Y10S514/879
摘要： Methods for the production of purified, catalytically active, recombinant memapsin 2 have been developed. The substrate and subsite specificity of the catalytically active enzyme have been determined. The substrate and subsite specificity information was used to design substrate analogs of the natural memapsin 2 substrate that can inhibit the function of memapsin 2. The substrate analogs are based on peptide sequences, shown to be related to the natural peptide substrates for memapsin 2. The substrate analogs contain at least one analog of an amide bond which is not capable of being cleaved by memapsin 2. Processes for the synthesis of two substrate analogues including isosteres at the sites of the critical amino acid residues were developed and the substrate analogues, OMR99-1 and OM99-2, were synthesized. OM99-2 is based on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with the Leu-Ala peptide bond substituted by a transition-state isostere hydroxyethylene group (FIG. 1). The inhibition constant of OM99-2 is 1.6×10−9 M against recombinant pro-memapsin 2. Crystallography of memapsin 2 bond to this inhibitor was used to determine the three dimensional structure of the protein, as well as the importance of the various residues in binding. This information can be used by those skilled in the art to design new inhibitors, using commercially available software programs and techniques familiar to those in organic chemistry and enzymology, to design new inhibitors to memapsin 2, useful in diagnostics and for the treatment and/or prevention of Alzheimer's disease.
摘要翻译：已经开发了用于生产纯化的，催化活性的重组突变蛋白2的方法。已经确定了催化活性酶的底物和亚位点特异性。底物和亚位点特异性信息用于设计可以抑制膜蛋白2功能的天然memapsin 2底物的底物类似物。底物类似物基于肽序列，显示与memapsin 2的天然肽底物相关。底物类似物含有至少一个酰胺键的类似物，该类似物不能被膜蛋白2切割。开发了两个底物类似物合成的关键氨基酸残基位点处的等位基因，底物类似物OMR99- 1和OM99-2。 OM99-2基于由过渡态等电位羟基亚乙基取代的Leu-Ala肽键的八肽Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe（SEQ ID NO：28）（图1 ）。 OM99-2的抑制常数为1.6×10-9M，与重组前胶原蛋白2相似。蛋白2的结构与该抑制剂结合使用，用于测定蛋白质的三维结构，以及各种残基的重要性绑定。本领域技术人员可以使用本信息来设计新的抑制剂，使用商业上可获得的有机化学和酶学方面熟悉的软件程序和技术来设计新的抑制剂2，可用于诊断和治疗和/或预防阿尔茨海默病。

3. 发明授权

US06545127B1 Catalytically active recombinant memapsin and methods of use thereof 失效
标题翻译：催化活性重组膜突触蛋白及其使用方法
公开(公告)号：US06545127B1
公开(公告)日：2003-04-08
申请号：US09604608
申请日：2000-06-27
申请人： Jordan J. N. Tang , Xinli Lin , Gerald Koelsch , Lin Hong
发明人： Jordan J. N. Tang , Xinli Lin , Gerald Koelsch , Lin Hong
IPC分类号： G01N3348
CPC分类号： C07K5/1021 , A61K38/00 , A61K39/00 , C07K1/1136 , C07K5/06026 , C07K5/06043 , C07K5/0806 , C07K2299/00 , C12N9/6421 , C12N9/6478 , Y02A90/26 , Y10S514/879
摘要： Methods for the production of purified, catalytically active, recombinant memapsin 2 have been developed. The substrate and subsite specificity of the catalytically active enzyme have been determined. The substrate and subsite specificity information was used to design substrate analogs of the natural memapsin 2 substrate that can inhibit the function of memapsin 2. The substrate analogs are based on peptide sequences, shown to be related to the natural peptide substrates for memapsin 2. The substrate analogs contain at least one analog of an amide bond which is not capable of being cleaved by memapsin 2. Processes for the synthesis of two substrate analogues including isosteres at the sites of the critical amino acid residues were developed and the substrate analogues, OMR99-1 and OM99-2, were synthesized. OM99-2 is based on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with the Leu-Ala peptide bond substituted by a transition-state isostere hydroxyethylene group (FIG. 1). The inhibition constant of OM99-2 is 1.6×10−9M against recombinant pro-memapsin 2. Crystallography of memapsin 2 bound to this inhibitor was used to determine the three dimensional structure of the protein, as well as the importance of the various residues in binding. This information can be used by those skilled in the art to design new inhibitors, using commercially available software programs and techniques familiar to those in organic chemistry and enzymology, to design new inhibitors to memapsin 2, useful in diagnostics and for the treatment and/or prevention of Alzheimer's disease.
摘要翻译：已经开发了用于生产纯化的，催化活性的重组突变蛋白2的方法。已经确定了催化活性酶的底物和亚位点特异性。底物和亚位点特异性信息用于设计可以抑制膜蛋白2功能的天然memapsin 2底物的底物类似物。底物类似物基于肽序列，显示与memapsin 2的天然肽底物相关。底物类似物含有至少一个酰胺键的类似物，该类似物不能被膜蛋白2切割。开发了两个底物类似物合成的关键氨基酸残基位点处的等位基因，底物类似物OMR99- 1和OM99-2。 OM99-2基于由过渡态等电位羟基亚乙基取代的Leu-Ala肽键的八肽Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe（SEQ ID NO：28）（图1 ）。 OM99-2的抑制常数为1.6×10 -9 M，与重组pro-memapsin2结合使用与此抑制剂结合的胶原蛋白2的结晶学，以确定蛋白质的三维结构，以及各种残基在结合中的重要性。本领域技术人员可以使用本信息来设计新的抑制剂，使用商业上可获得的有机化学和酶学方面熟悉的软件程序和技术来设计新的抑制剂2，可用于诊断和治疗和/或预防阿尔茨海默病。

4. 发明授权

US06303380B1 Construction of retroviral producer cells from adenoviral and retroviral vectors 有权
标题翻译：从腺病毒和逆转录病毒载体构建逆转录病毒生产细胞
公开(公告)号：US06303380B1
公开(公告)日：2001-10-16
申请号：US09301846
申请日：1999-04-29
申请人： Xinli Lin , Jordan J. N. Tang
发明人： Xinli Lin , Jordan J. N. Tang
IPC分类号： C12N510
CPC分类号： C12N15/86 , C12N2710/10344 , C12N2740/13043 , C12N2740/13052
摘要： A combination of adenoviral and retroviral vectors used to construct second generation packaging cells that deliver marker genes to target cells is described. A vector based upon Moloney murine leukemia virus (MLV) was used to deliver marker genes, and an adenovirus-based delivery system was used to deliver MLV structural genes (gagpol and env) to cultured cells. The procedure transformed the cells into new retroviral producer cells, which generate replication-incompetent retroviral particles in the culture supernatant for transferring marker genes to target cells. The titer of the retroviral-containing supernatant generated from the second generation producer cells reached above 105 cfu/ml which is comparable to the MLV-based producer cell lines currently used in human gene therapy trials. The vector and procedures are adaptable for experimental human gene therapy in which the new producer cells are transplanted into patients for continuous gene transfer.
摘要翻译：描述了用于构建将标记基因递送至靶细胞的第二代包装细胞的腺病毒和逆转录病毒载体的组合。使用基于莫洛尼鼠白血病病毒（MLV）的载体递送标记基因，并且使用基于腺病毒的递送系统将MLV结构基因（gagpol和env）递送至培养的细胞。该过程将细胞转化为新的逆转录病毒生产细胞，其在培养上清液中产生复制不足的逆转录病毒颗粒，用于将标记基因转移至靶细胞。从第二代生产细胞产生的含有逆转录病毒的上清液的滴度高于105cfu / ml，这与目前用于人类基因治疗试验的基于MLV的生产细胞系相当。载体和程序适用于实验性人类基因治疗，其中新的生产细胞被移植到患者中用于连续基因转移。

5. 发明申请

US20070218535A1 Methods for production of recombinant alpha1-antitrypsin 审中-公开
标题翻译：生产重组α1-抗胰蛋白酶的方法
公开(公告)号：US20070218535A1
公开(公告)日：2007-09-20
申请号：US11605619
申请日：2006-11-28
申请人： Xinli Lin , Daniel Medynski
发明人： Xinli Lin , Daniel Medynski
IPC分类号： C12N9/64
CPC分类号： C07K14/8125 , C12N9/99
摘要： Methods of producing properly folded recombinant α1-antitrypsin (AAT) polypeptide are provided. Denatured recombinant AAT polypeptide is refolded by first solubilizing the polypeptide with a chaotroph at high pH, followed by refolding in the presence of reduced concentrations of chaotroph and in the presence of PEG, glycerol or sucrose, or a detergent while the pH is slowly reduced and is generally maintained.
摘要翻译：提供了生产正确折叠的重组α1-抗胰蛋白酶（AAT）多肽的方法。变性重组AAT多肽通过首先在高pH下溶解具有混沌营养素的多肽，然后在浓度较低的混合营养物存在下和在PEG，甘油或蔗糖或洗涤剂的存在下重折叠，同时pH缓慢降低，一般维持。

6. 发明申请

US20120165268A1 P53 FUSION PROTEINS AND METHODS OF MAKING AND USING THEREOF 审中-公开
标题翻译： P53融合蛋白及其制备及其使用方法
公开(公告)号：US20120165268A1
公开(公告)日：2012-06-28
申请号：US13321805
申请日：2009-05-28
申请人： Xinli Lin , Michelle Lafevre-Bernt
发明人： Xinli Lin , Michelle Lafevre-Bernt
IPC分类号： A61K38/17 , C07K14/47 , C07K1/00 , A61P35/00 , C07K1/18 , C07K1/20 , C07K1/22 , C07K1/16 , C07K19/00 , C07K1/14
CPC分类号： C07K14/4746
摘要： Biologically active tetrameric p53 proteins and p53 fusion proteins are provided. These proteins may be generated and refolded into tetrameric form using denatured proteins produced from E. coli. Therapeutic uses of p53 proteins and p53 fusion proteins are also provided.
摘要翻译：提供生物活性四聚体p53蛋白和p53融合蛋白。这些蛋白质可以使用从大肠杆菌产生的变性蛋白质产生并重折叠成四聚体形式。还提供了p53蛋白和p53融合蛋白的治疗用途。

7. 发明申请

US20070009506A1 Methods for production of recombinant urokinase 审中-公开
标题翻译：重组尿激酶生产方法
公开(公告)号：US20070009506A1
公开(公告)日：2007-01-11
申请号：US11444594
申请日：2006-05-31
申请人： Xinli Lin
发明人： Xinli Lin
IPC分类号： A61K38/48 , C12N9/72
CPC分类号： C12N9/6462 , C12Y304/21073
摘要： Highly efficient methods of producing properly folded recombinant urokinase are provided. Denatured recombinant pro-urokinase is refolded by first solubilizing the protein with a chaotroph at high pH, followed by refolding in the presence of reduced concentrations of chaotroph while the pH is slowly reduced.
摘要翻译：提供了生产正确折叠的重组尿激酶的高效方法。变性重组原尿激酶通过首先在高pH下溶解具有混沌营养蛋白的蛋白质重新折叠，随后在减少浓度的混合营养物的存在下重折叠，同时pH缓慢降低。

8. 发明申请

US20060287504A1 Universal procedure for refolding recombinant proteins 审中-公开
公开(公告)号：US20060287504A1
公开(公告)日：2006-12-21
申请号：US11506739
申请日：2006-08-18
申请人： Xinli Lin
发明人： Xinli Lin
IPC分类号： C12P21/06 , C07K14/47
CPC分类号： C12N9/6478 , A61K38/00 , A61K39/00 , C07K1/1133 , C07K1/1136 , C07K5/1021 , C12N9/6421
摘要： A universal folding method that has been demonstrated to be effective in refolding a variety of very different proteins expressed in bacteria as inclusion bodies has been developed. Representative proteins that can be dissolved and refolded in biologically active form, with the native structure, are shown in Table I. The method has two key steps to unfold and then refold the proteins expressed in the inclusion bodies. The first step is to raise the pH of the protein solution in the presence of denaturing agents to pH greater than 9, preferably 10. The protein solution may be maintained at the elevated pH for a period of up to about 24 hours, or the pH immediately decreased slowly, in increments of about 0.2 pH units/24 hours, until the solution reaches a pH of about 8.0, or both steps used. In the preferred embodiment, purified inclusion bodies are dissolved in 8 M urea, 0.1 M Tris, 1 mM glycine, 1 mM EDTA, 10 mM beta-mercaptoethanol, 10 mM dithiothreitol (DTT), 1 mM redued glutathion (GSH), 0.1 mM oxidized glutathion (GSSG), pH 10. The absorbance at 280 nm (OD280) of the protein solution is 5.0. This solution is rapidly diluted into 20 volumes of 20 mM Tris base. The resulting solutin is adjusted to pH 9.0 with 1 M HCl and is kept at 4° C. for 24 hr. The pH is adjusted to pH 8.8 and the solution is kept at 4° C. for another 24 hrs. This process is repeated until the pH is adjusted to 8.0. After 24 hr at pH 8.0, the refolded proteins can be concentrated by ultrafiltration and applied to a gel filtration column for purification.

9. 发明授权

US06583268B2 Universal procedure for refolding recombinant proteins 失效
公开(公告)号：US06583268B2
公开(公告)日：2003-06-24
申请号：US09752878
申请日：2000-12-28
申请人： Xinli Lin
发明人： Xinli Lin
IPC分类号： C07K1400
CPC分类号： C12N9/6478 , A61K38/00 , A61K39/00 , C07K1/1133 , C07K1/1136 , C07K5/1021 , C12N9/6421
摘要： A universal folding method that has been demonstrated to be effective in refolding a variety of very different proteins expressed in bacteria as inclusion bodies has been developed. Representative proteins that can be dissolved and refolded in biologically active form, with the native structure, are shown in Table I. The method has two key steps to unfold and then refold the proteins expressed in the inclusion bodies. The first step is to raise the pH of the protein solution in the presence of denaturing agents to pH greater than 9, preferably 10. The protein solution may be maintained at the elevated pH for a period of up to about 24 hours, or the pH immediately decreased slowly, in increments of about 0.2 pH units/24 hours, until the solution reaches a pH of about 8.0, or both steps used. In the preferred embodiment, purified inclusion bodies are dissolved in 8 M urea, 0.1 M Tris, 1 mM glycine, 1 mM EDTA, 10 mM beta-mercaptoethanol, 10 mM dithiothreitol (DTT), 1 mM redued glutathion (GSH), 0.1 mM oxidized glutathion (GSSG), pH 10. The absorbance at 280 nm (OD280) of the protein solution is 5.0. This solution is rapidly diluted into 20 volumes of 20 mM Tris base. The resulting solutin is adjusted to pH 9.0 with 1 M HCl and is kept at 4° C. for 24 hr. The pH is adjusted to pH 8.8 and the solution is kept at 4° C. for another 24 hrs. This process is repeated until the pH is adjusted to 8.0. After 24 hr at pH 8.0, the refolded proteins can be concentrated by ultrafiltration and applied to a gel filtration column for purification.

10. 发明授权

US06225103B1 Cloning and characterization of napsin 失效
标题翻译： napsin的克隆和表征
公开(公告)号：US06225103B1
公开(公告)日：2001-05-01
申请号：US08974691
申请日：1997-11-20
申请人： Gerald Keolsch , Xinli Lin , Jordan Tang
发明人： Gerald Keolsch , Xinli Lin , Jordan Tang
IPC分类号： A61K3857
CPC分类号： C12N9/6478
摘要： A previously unknown aspartic protease capable of cleavage of proteins by hydrolysis, referred to herein as “napsin”, has been cloned from a human liver library. Two cDNA clones have been cloned, sequenced and expressed. These encode isozymes of the protease, referred to as “napsin A” and “napsin B”. The gene has also be obtained and partially sequenced. A process for rapid purification of the enzyme using immobilized petpstatin has also been developed, and enzyme isolated from human kidney tissue. Polyclonal antibodies to the enzymes have been made which are also useful for isolation and detection of the enzyme. Similarities to other aspartic proteases, especially cathepsin D, establish the usefulness of the enzyme in diagnostic assays as well as as a protease. Either or both the amount or type of napsin expressed in a particular tissue can be determined using labelled antibodies or nucleotide probes to the napsin.
摘要翻译：已经从人肝脏文库克隆了能够通过水解裂解蛋白质的以前未知的天冬氨酸蛋白酶，这里称为“重叠蛋白酶”（Napsin））。已经克隆，测序和表达了两个cDNA克隆。这些编码蛋白酶的同功酶，称为“napsin A”和“napsin B”。该基因也可获得并部分测序。还开发了使用固定的petpstatin快速纯化酶的方法，并从人肾组织中分离酶。已经制备了对酶的多克隆抗体，其也可用于分离和检测酶。与其他天冬氨酸蛋白酶，特别是组织蛋白酶D的相似性确定了酶在诊断测定中的用途以及作为蛋白酶。在特定组织中表达的napsin的量或类型中的任一个或两者可以使用标记的抗体或对napsin的核苷酸探针来确定。

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