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    UDP-galactose:beta-N-acetyl-glucosamine beta-1,3-galactosyl transferases, beta-3Gal-T5
    1.
    发明申请
    UDP-galactose:beta-N-acetyl-glucosamine beta-1,3-galactosyl transferases, beta-3Gal-T5 失效
    UDP-半乳糖：β-N-乙酰基 - 葡糖胺β-1,3-半乳糖基转移酶，β-3Gal-T5

    公开(公告)号：US20060099699A1

    公开(公告)日：2006-05-11

    申请号：US11305413

    申请日：2005-12-16

    申请人： Henrik Clausen ,  Margarida Amado

    发明人： Henrik Clausen ,  Margarida Amado

    IPC分类号： C12N9/10 ,  C07H21/04 ,  C08B37/00 ,  C12P21/06

    CPC分类号： C12N9/1051 ,  C07K2319/00

    摘要： A novel gene defining a novel enzyme in the UDP-D-galactose: β-N-acetylglucosamine/β-N-acetylgalactosamine β1,3galactosyltransferase family, termed β3Gal-T5, with unique enzymatic properties is disclosed. The enzymatic activity of β3Gal-T5 is shown to be distinct from that of previously identified enzymes of this gene family. The invention discloses isolated DNA molecules and DNA constructs encoding β3Gal-T5 and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting β3Gal-T5 activity, as well as cloning and expression vectors including such DNA, cells transfected with the vectors, and recombinant methods for providing β3Gal-T5. The enzyme β3Gal-T5 and β3Gal-T5-active derivatives thereof are disclosed, in particular soluble derivatives comprising the catalytically active domain of β3Gal-T5. Further, the invention discloses methods of obtaining β1,3galactosyl glycosylated saccharides, glycopeptides or glycoproteins by use of an enzymatically active β3Gal-T5 protein or, fusion protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active β3Gal-T5 protein as an expression system for recombinant production of such glycopeptides or glycoproteins. Also a method for the identification of DNA sequence variations in the β3Gal-T5 gene by isolating DNA from a patient, amplifying β3Gal-T5-coding exons by PCR, and detecting the presence of DNA sequence variation, are disclosed.

    摘要翻译： 公开了一种在UDP-D-半乳糖：β-N-乙酰氨基葡萄糖/β-N-乙酰半乳糖胺β1,3半乳糖转移酶家族中定义新型酶的新基因，称为β3Gal-T5，具有独特的酶学性质。 β3Gal-T5的酶活性显示与以前鉴定的该基因家族酶的活性不同。 本发明公开了通过氨基酸缺失，取代或插入显示β3Gal-T5活性的编码β3Gal-T5及其衍生物的分离的DNA分子和DNA构建体，以及包括这种DNA的克隆和表达载体，用载体转染的细胞，以及 提供beta3Gal-T5的重组方法。 公开了酶β3Gal-T5和β3Gal-T5-活性衍生物，特别是包含β3Gal-T5的催化活性结构域的可溶性衍生物。 此外，本发明公开了通过使用酶活性β3Gal-T5蛋白或其融合蛋白或通过使用包含编码酶活性β3Gal-T5的DNA的载体稳定转染的细胞获得β1,3半乳糖基糖基化糖，糖肽或糖蛋白的方法 蛋白质作为用于重组生产这种糖肽或糖蛋白的表达系统。 还公开了通过从患者中分离DNA，通过PCR扩增β3Gal-T5编码外显子并检测DNA序列变异的存在来鉴定β3Gal-T5基因中的DNA序列变异的方法。

    UDP-galactose: β-N-acetyl-glucosamine β1,3 galactosyltransferases, β3gal-T5
    2.
    发明授权
    UDP-galactose: β-N-acetyl-glucosamine β1,3 galactosyltransferases, β3gal-T5 失效
    UDP-半乳糖：β-N-乙酰基 - 葡糖胺β1,3半乳糖基转移酶，β3gal-T5

    公开(公告)号：US07332279B2

    公开(公告)日：2008-02-19

    申请号：US10777828

    申请日：2004-02-12

    申请人： Henrik Clausen ,  Margarida Amado

    发明人： Henrik Clausen ,  Margarida Amado

    IPC分类号： C12Q1/68 ,  C12P21/06 ,  C12N9/10 ,  C12N1/20 ,  C12N15/70 ,  C07H21/04 ,  C07K1/00

    CPC分类号： C12N9/1051 ,  C07K2319/00

    摘要： A novel gene defining a novel enzyme in the UDP-D-galactose: beta-N-acetylglucosamine/beta-N-acetylgalactosamine beta 1,3galactosyltransferase family, termed beta3Gal-T5, with unique enzymatic properties is disclosed. The enzymatic activity of beta3Gal-T5 is shown to be distinct from that of previously identified enzymes of this gene family. The invention discloses isolated DNA molecules and DNA constructs encoding beta3Gal-T5 and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting beta3Gal-T5 activity, as well as cloning and expression vectors including such DNA, cells transfected with the vectors, and recombinant methods for providing beta3Gal-T5. The enzyme beta3Gal-T5 and beta3Gal-T5-active derivatives thereof are disclosed, in particular soluble derivatives comprising the catalytically active domain of beta3Gal-T5. Further, the invention discloses methods of obtaining beta 1,3galactosyl glycosylated saccharides, glycopeptides or glycoproteins by use of an enzymically active beta3Gal-T5 protein or fusion protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active beta3Gal-T5 protein as an expression system for recombinant production of such glycopeptides or glycoproteins. Also a method for the identification of DNA sequence variations in the beta3Gal-T5 gene by isolating DNA from a patient, amplifying beta3Gal-T5-coding exons by PCR, and detecting the presence of DNA sequence variation, are disclosed.

    摘要翻译： 公开了一种在UDP-D-半乳糖：β-N-乙酰氨基葡萄糖/β-N-乙酰半乳糖胺β1,3-半乳糖转移酶家族中定义新型酶的新基因，称为β3Gal-T5，具有独特的酶学性质。 β3Gal-T5的酶活性显示与以前鉴定的该基因家族酶的活性不同。 本发明公开了通过氨基酸缺失，取代或插入显示β3Gal-T5活性的编码β3Gal-T5及其衍生物的分离的DNA分子和DNA构建体，以及包括这种DNA的克隆和表达载体，用载体转染的细胞，以及 提供beta3Gal-T5的重组方法。 公开了酶β3Gal-T5和β3Gal-T5-活性衍生物，特别是包含β3Gal-T5的催化活性结构域的可溶性衍生物。 此外，本发明公开了通过使用酶活性β3Gal-T5蛋白或其融合蛋白或通过使用包含编码酶活性β3Gal-T5的DNA的载体稳定转染的细胞获得β1,3-半乳糖基糖基化糖，糖肽或糖蛋白的方法 蛋白质作为用于重组生产这种糖肽或糖蛋白的表达系统。 还公开了通过从患者中分离DNA，通过PCR扩增β3Gal-T5编码外显子并检测DNA序列变异的存在来鉴定β3Gal-T5基因中的DNA序列变异的方法。

    UDP-galactose:β-N-acetyl-glucosamine β-1,3-galactosyl transferases, β-3Gal-T5
    3.
    发明授权
    UDP-galactose:β-N-acetyl-glucosamine β-1,3-galactosyl transferases, β-3Gal-T5 失效
    UDP-半乳糖：β-N-乙酰基 - 葡糖胺β-1,3-半乳糖基转移酶，β-3Gal-T5

    公开(公告)号：US07476527B2

    公开(公告)日：2009-01-13

    申请号：US11305413

    申请日：2005-12-16

    申请人： Henrik Clausen ,  Margarida Amado

    发明人： Henrik Clausen ,  Margarida Amado

    IPC分类号： C12N9/10 ,  C12N15/00 ,  C12Q1/00 ,  C12Q1/68 ,  C12P21/04 ,  C07H21/04 ,  C07K1/00

    CPC分类号： C12N9/1051 ,  C07K2319/00

    摘要： A novel gene defining a novel enzyme in the UDP-D-galactose: β-N-acetylglucosamine / β-N-acetylgalactosamine β1,3galactosyltransferase family, termed β3Gal-T5, with unique enzymatic properties is disclosed. The enzymatic activity of β3Gal-T5 is shown to be distinct from that of previously identified enzymes of this gene family. The invention discloses isolated DNA molecules and DNA constructs encoding β3Gal-T5 and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting β3Gal-T5 activity, as well as cloning and expression vectors including such DNA, cells transfected with the vectors, and recombinant methods for providing β3Gal-T5. The enzyme β3Gal-T5 and β3Gal-T5-active derivatives thereof are disclosed, in particular soluble derivatives comprising the catalytically active domain of β3Gal-T5. Further, the invention discloses methods of obtaining β1,3galactosyl glycosylated saccharides, glycopeptides or glycoproteins by use of an enzymatically active β3Gal-T5protein or, fusion protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active β3Gal-T5 protein as an expression system for recombinant production of such glycopeptides or glycoproteins. Also a method for the identification of DNA sequence variations in the β3Gal-T5 gene by isolating DNA from a patient, amplifying β3Gal-T5-coding exons by PCR, and detecting the presence of DNA sequence variation, are disclosed.

    摘要翻译： 公开了一种在UDP-D-半乳糖：β-N-乙酰氨基葡萄糖/β-N-乙酰半乳糖胺β1,3半乳糖转移酶家族中定义新型酶的新基因，称为β3Gal-T5，具有独特的酶学性质。 β3Gal-T5的酶活性显示与以前鉴定的该基因家族酶的活性不同。 本发明公开了通过氨基酸缺失，取代或插入显示β3Gal-T5活性的编码β3Gal-T5及其衍生物的分离的DNA分子和DNA构建体，以及包括这种DNA的克隆和表达载体，用载体转染的细胞，以及 提供beta3Gal-T5的重组方法。 公开了酶β3Gal-T5和β3Gal-T5-活性衍生物，特别是包含β3Gal-T5的催化活性结构域的可溶性衍生物。 此外，本发明公开了通过使用酶活性β3Gal-T5蛋白或其融合蛋白或通过使用包含编码酶活性β3Gal-T5蛋白的DNA的载体稳定转染的细胞获得β1,3半乳糖苷糖基化糖，糖肽或糖蛋白的方法 作为重组生产这种糖肽或糖蛋白的表达系统。 还公开了通过从患者中分离DNA，通过PCR扩增β3Gal-T5编码外显子并检测DNA序列变异的存在来鉴定β3Gal-T5基因中的DNA序列变异的方法。

    UDP-galactose: &bgr;-N-acetyl-glucosamine &bgr;1,3galactosyltransferases, &bgr;3Gal-T5
    4.
    发明授权
    UDP-galactose: &bgr;-N-acetyl-glucosamine &bgr;1,3galactosyltransferases, &bgr;3Gal-T5 失效
    UDP-半乳糖：β-N-乙酰葡糖胺β1,3半乳糖转移酶，β3Gal-T5

    公开(公告)号：US06800468B1

    公开(公告)日：2004-10-05

    申请号：US09831630

    申请日：2001-05-10

    申请人： Henrik Clausen ,  Margarida Amado

    发明人： Henrik Clausen ,  Margarida Amado

    IPC分类号： C12N1509

    CPC分类号： C12N9/1051 ,  C07K2319/00

    摘要： A novel gene defining a novel enzyme in the UDP-D-galactose: &bgr;-N-acetylglucosamine/&bgr;-N-acetylgalactosamine &bgr;1,3galactosyltransferase family, termed &bgr;3Gal-T5, with unique enzymatic properties is disclosed. The enzymatic activity of &bgr;3Gal-T5 is shown to be distinct from that of previously identified enzymes of this gene family. The invention discloses isolated DNA molecules and DNA constructs encoding &bgr;3Gal-T5 and derivatives thereof by way of amino acid deletion, substitution or insertion exhibiting &bgr;3Gal-T5 activity, as well as cloning and expression vectors including such DNA, cells tranfected with the vectors, and recombinant methods for providing &bgr;3Gal-T5. The enzyme &bgr;3Gal-T5 and &bgr;3Gal-T5-active derivatives thereof are disclosed, in particular soluble derivatives comprising the catalytically active domain of &bgr;3Gal-T5. Further, the invention discloses methods of obtaining &bgr;1,3galactosyl glycosylated saccharides, glycopeptides or glycoproteins by use of an enzymically active &bgr;3Gal-T5 protein or fusion protein thereof or by using cells stably transfected with a vector including DNA encoding an enzymatically active &bgr;3Gal-T5 protein as an expression system for recombinant production of such glycopeptides or glycoproteins. Also a method for the identification of DNA sequence variations in the &bgr;3Gal-T5 gene by isolating DNA from a patient, amplifying &bgr;3Gal-T5-coding exons by PCR, and detecting the presence of DNA sequence variation, are disclosed.

    摘要翻译： 公开了一种在UDP-D-半乳糖：β-N-乙酰氨基葡萄糖/β-N-乙酰半乳糖胺β1,3半乳糖转移酶家族中定义新型酶的新基因，称为β3Gal-T5，具有独特的酶学性质。 β3Gal-T5的酶活性显示与以前鉴定的该基因家族酶的活性不同。 本发明公开了通过氨基酸缺失，取代或插入显示β3Gal-T5活性的编码β3Gal-T5及其衍生物的分离的DNA分子和DNA构建体，以及包含这些DNA的克隆和表达载体，用载体转染的细胞，以及 提供beta3Gal-T5的重组方法。 公开了酶β3Gal-T5和β3Gal-T5-活性衍生物，特别是包含β3Gal-T5的催化活性结构域的可溶性衍生物。 此外，本发明公开了通过使用酶活性β3Gal-T5蛋白或其融合蛋白或通过使用包含编码酶活性β3Gal-T5蛋白的DNA的载体稳定转染的细胞获得β1,3半乳糖基糖基化糖，糖肽或糖蛋白的方法 作为重组生产这种糖肽或糖蛋白的表达系统。 还公开了通过从患者中分离DNA，通过PCR扩增β3Gal-T5编码外显子并检测DNA序列变异的存在来鉴定β3Gal-T5基因中的DNA序列变异的方法。

    Methods for glyco-engineering plant cells for controlled human O-glycosylation
    6.
    发明授权
    Methods for glyco-engineering plant cells for controlled human O-glycosylation 有权
    糖酵解植物细胞用于受控人O-糖基化的方法

    公开(公告)号：US09024110B2

    公开(公告)日：2015-05-05

    申请号：US13070248

    申请日：2011-03-23

    申请人： Zhang Yang ,  Damian Paul Drew ,  Emma Adhiambo Arigi ,  Peter Ulvskov ,  Steven B. Levery ,  Eric Bennett ,  Henrik Clausen ,  Brent Larsen Petersen

    发明人： Zhang Yang ,  Damian Paul Drew ,  Emma Adhiambo Arigi ,  Peter Ulvskov ,  Steven B. Levery ,  Eric Bennett ,  Henrik Clausen ,  Brent Larsen Petersen

    IPC分类号： C12N15/82 ,  C12N15/12 ,  C12N15/30 ,  C12N15/31 ,  C12N9/10 ,  C12N9/90 ,  C12P19/00 ,  C12P19/44 ,  A61K38/47 ,  C12N1/13 ,  C07K14/47 ,  C12P21/00

    CPC分类号： C12N15/8257 ,  C07K14/4727 ,  C12N15/8245 ,  C12P21/005

    摘要： This invention discloses the development of a novel platform for recombinant production of bioactive glycoproteins and cancer specific vaccines in plants. Plants and plant cell cultures have been humanized with respect to human mucin-type protein O-glycosylation. A panel of plant cell factories for production of recombinant glycoproteins with designed human O-glycosylation, including an improved cancer vaccine candidate, has been developed. The platform provides basis for i) production of an essentially unlimited array of O-glycosylated human glycoprotein therapeutics, such as human interferon α2B and podoplanin, and ii) for further engineering of additional cancer specific O-glycans on glycoproteins of therapeutical value. Currently, mammalian cells are required for human O-glycosylation, but plants offer a unique cell platform for engineering O-glycosylation since they do not perform human type O-glycosylation. Introduction of O-glycosylation into plant cells requires i) that wild-type plant cells do not modify the target peptide substrates and ii) that the appropriate enzymes and substrates are introduced into of plant cells such that O-glycosylation in the secretory pathway proceed and the glycosylated peptide substrates are preferentially exported to the exterior of the cell or accumulated in the cell. In this invention i) the integrity of transiently and stably expressed ‘mucin’ type target peptides in plants cells has been determined and ii) mucin-type O-glycosylation has been established in plants by transient and stable introduction of a Pseudomonas aeruginosa C4-epimerase, the human polypeptide GalNAc-transferases T2 and T4 (GalNAc-T2 and T4) and various human target peptides or proteins. In the present invention GalNAc-T2 and -T4 have been used to produce a Tn cancer glycoform of MUC1.

    摘要翻译： 本发明公开了在植物中重组生物活性糖蛋白和癌特异性疫苗重组生产的新平台的开发。 植物和植物细胞培养物相对于人粘蛋白型蛋白O-糖基化已被人源化。 已经开发了一组用于生产具有设计的人O-糖基化的重组糖蛋白的植物细胞工厂，包括改进的癌症疫苗候选物。 该平台为i）生产基本上无限量的O-糖基化的人类糖蛋白治疗剂例如人干扰素α2B和podoplanin的基础，以及ii）用于在治疗价值的糖蛋白上进一步工程化另外的癌症特异性O-聚糖。 目前，哺乳动物细胞是人O-糖基化所必需的，但由于它们不进行人类O-糖基化，植物提供了独特的用于工程化O-糖基化的细胞平台。 将O-糖基化引入到植物细胞中需要i）野生型植物细胞不修饰靶肽底物，和ii）将合适的酶和底物引入植物细胞，使分泌途径中的O-糖基化进行， 糖基化肽底物优选地输出到细胞的外部或者积聚在细胞中。 在本发明中，i）已经确定了植物细胞中瞬时稳定表达的“粘蛋白”型靶肽的完整性，并且ii）通过暂时稳定地引入铜绿假单胞菌C4差向异构酶，在植物中建立了粘蛋白型O-糖基化 ，人多肽GalNAc-转移酶T2和T4（GalNAc-T2和T4）和各种人靶肽或蛋白质。 在本发明中，已经使用GalNAc-T2和-T4来产生MUC1的Tn癌糖蛋白。

    Glycopegylated interferon α
    8.
    发明授权
    Glycopegylated interferon α 有权
    糖基化干扰素α

    公开(公告)号：US08268967B2

    公开(公告)日：2012-09-18

    申请号：US11659942

    申请日：2005-09-12

    申请人： Shawn DeFrees ,  David A. Zopf ,  Henrik Clausen ,  Ruye Xing

    发明人： Shawn DeFrees ,  David A. Zopf ,  Henrik Clausen ,  Ruye Xing

    IPC分类号： C07K14/56 ,  A61K38/21

    CPC分类号： A61K38/212 ,  A61K47/60 ,  Y02A50/463 ,  Y02A50/473

    摘要： The present invention provides IFN-α conjugates including IFN-α peptides and modifying groups such as PEG moieties. The IFN-α peptide and modifying group are linked via an intact glycosyl linking group interposed between and covalently attached to the IFN-α peptide and the modifying group. The IFN-α conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar onto an amino acid or a glycosyl residue on the IFN-α peptide. Also provided are methods for preparing the IFN-α conjugates, methods for treating various disease conditions with the IFN-α conjugates, and pharmaceutical formulations including the IFN-α conjugates.

    摘要翻译： 本发明提供IFN-α缀合物，包括IFN-α肽和修饰基团例如PEG部分。 IFN-α肽和修饰基团通过介于IFN-α肽和修饰基团之间并共价连接的完整糖基连接基团连接。 通过糖基转移酶的作用由糖基化肽形成IFN-α缀合物。 糖基转移酶将修饰的糖连接到IFN-α肽上的氨基酸或糖基残基上。 还提供了制备IFN-α缀合物的方法，用IFN-α缀合物治疗各种疾病状况的方法，以及包括IFN-α缀合物的药物制剂。

    Enzymatic Conversion Of Blood Group A, B, And AB Red Blood Cells Using Alpha-N-Acetylgalactosaminidases and Alpha-Galactosidases With Unique Substrate Specificities And Kinetic Properties
    9.
    发明申请
    Enzymatic Conversion Of Blood Group A, B, And AB Red Blood Cells Using Alpha-N-Acetylgalactosaminidases and Alpha-Galactosidases With Unique Substrate Specificities And Kinetic Properties 有权
    使用具有独特底物特异性和动力学性质的α-N-乙酰半乳糖胺酶和α-半乳糖苷酶对A组，B组和AB组血液细胞进行酶转化

    公开(公告)号：US20120202273A1

    公开(公告)日：2012-08-09

    申请号：US13205588

    申请日：2011-08-08

    申请人： Henrik Clausen ,  Humberto de la Vega ,  Chery Hill ,  Qiyong Peter Liu

    发明人： Henrik Clausen ,  Humberto de la Vega ,  Chery Hill ,  Qiyong Peter Liu

    IPC分类号： C12N1/20

    CPC分类号： C12N5/0641 ,  C12N9/2402 ,  C12N9/2465 ,  C12Q1/34 ,  C12R1/465 ,  C12Y302/01022 ,  C12Y302/01049

    摘要： This invention relates to enzymatic removal of type A and B antigens from blood group A, B, and AB reactive cells in blood products, and thereby converting these to non-A and non-B reactive cells. The invention further relates to using unique α-N-acetylgalactosaminidases and α-galactosidases with superior kinetic properties for removing the immunodominant monosaccharides of the blood group A and B antigens and improved performance in enzymatic conversion of red blood cells. The preferred unique α-N-acetylgalactosaminidases and α-galactosidases exhibit the following characteristics: (i) exclusive, preferred or no less than 10% substrate specificity for the type A and B branched polysaccharide structures relative to measurable activity with simple mono- and disaccharide structures and aglycon derivatives hereof; (ii) optimal performance at neutral pH with blood group oligosaccharides and in enzymatic conversion of cells; and (iii) a favorable kinetic constant Km with mono- and oligosaccharide substrates. The conversion methods of the invention use significantly lower amounts of recombinant glycosidase enzymes than previous and result in complete sero-conversion of all blood group A and B red cells.

    摘要翻译： 本发明涉及从血型A，B，AB型反应性细胞中分离A型和B型抗原，从而将其转化为非A型和非B型细胞。 本发明还涉及使用独特的α-N-乙酰半乳糖胺酶和具有优异动力学性质的α-半乳糖苷酶，用于去除血型A和B抗原的免疫优势单糖，并提高了红细胞酶促转化的表现。 优选的独特的α-N-乙酰半乳糖胺酶和α-半乳糖苷酶具有以下特征：（i）相对于简单的单糖和二糖的可测量的活性，对A型和B型支链多糖结构的排列，优选或不低于10％的底物特异性 结构和糖苷配基衍生物; （ii）在中性pH下用血型寡糖和细胞酶促转化的最佳性能; 和（iii）具有单糖和寡糖底物的有利的动力学常数Km。 本发明的转化方法比以前使用显着更低量的重组糖苷酶，并导致所有血型A和B红细胞的完全血清转化。