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    Probiotic microorganisms producing chimeric human growth hormone fused with Fc fragment of human IgG for oral delivery system and methods for producing them
    5.
    发明申请
    Probiotic microorganisms producing chimeric human growth hormone fused with Fc fragment of human IgG for oral delivery system and methods for producing them 审中-公开
    与用于口服递送系统的人IgG的Fc片段融合的产生嵌合人生长激素的益生菌微生物及其制备方法

    公开(公告)号：US20060094083A1

    公开(公告)日：2006-05-04

    申请号：US11211464

    申请日：2005-08-26

    申请人： Yun-Jaie Choi ,  Chang-Hoon Lee ,  Jung-Hee Woo ,  Seung-Ha Kang ,  Seung-Hoon Choi ,  Sang-Kee Kang

    发明人： Yun-Jaie Choi ,  Chang-Hoon Lee ,  Jung-Hee Woo ,  Seung-Ha Kang ,  Seung-Hoon Choi ,  Sang-Kee Kang

    IPC分类号： C12P21/06 ,  C12N1/21 ,  C12N1/18 ,  C07K14/61

    CPC分类号： C07K14/61 ,  C07K2319/30 ,  C12N15/746 ,  C12N15/815

    摘要： The present invention relates to probiotic microorganisms producing chimeric human growth hormone for oral use and methods for preparing them. The invention provides probiotic Lactobacillus or yeast transformant expressing chimeric protein which is human growth hormone fused with Fc fragment of human IgG, in which the transformants are safely delivered into intestine though oral route. Also, the invention provides a chimeric protein-expressing vector which can induce transcytosis in intestine epithelial cells. Accordingly, the invention demonstrates that the chimeric protein for oral delivery system can be absorbed in intestine, and delivery of the chimeric protein by oral route using Lactobacillus has very excellent efficiency in vivo test in rats. Accordingly, the Lactobacillus of the present invention is an excellent deliverer of protein drugs.

    摘要翻译： 本发明涉及产生用于口服的嵌合人生长激素的益生菌微生物及其制备方法。 本发明提供了表达嵌合蛋白的益生菌乳杆菌或酵母转化体，其是与人IgG的Fc片段融合的人生长激素，其中转化体通过口服途径安全递送至肠。 此外，本发明提供了可诱导肠上皮细胞转胞吞作用的嵌合蛋白质表达载体。 因此，本发明表明用于口服递送系统的嵌合蛋白质可以在肠中被吸收，并且使用乳杆菌通过口服途径递送嵌合蛋白质具有非常优异的大鼠体内试验效率。 因此，本发明的乳杆菌是蛋白质药物的优良递送剂。

    Methods for expression and purification of immunotoxins
    7.
    发明授权
    Methods for expression and purification of immunotoxins 有权
    免疫毒素的表达和纯化方法

    公开(公告)号：US07892786B2

    公开(公告)日：2011-02-22

    申请号：US10566886

    申请日：2004-08-02

    申请人： David M. Neville ,  Jung-Hee Woo ,  Yuan-Yi Liu

    发明人： David M. Neville ,  Jung-Hee Woo ,  Yuan-Yi Liu

    IPC分类号： C12N15/09 ,  C12N15/10 ,  C12N15/81

    CPC分类号： C07K1/18 ,  A61K47/6829 ,  A61K47/6849 ,  C07K16/2809 ,  C07K2317/622 ,  C07K2319/00 ,  C12N1/16

    摘要： The present invention relates to a method of expressing an immunotoxin in Pichia pastoris strain mutated to toxin resistance comprising a) growing the Pichia pastoris in a growth medium comprising an enzymatic digest of protein and yeast extract and maintaining a dissolved oxygen concentration at 40% and above; and b) performing methanol induction with a limited methanol feed of 0.5-0.75 ml/min/IO L of initial volume during induction along with a continuous infusion of yeast extract at a temperature below 17.5° C., antifoaming agent supplied up to 0.07%, agitation reduced to 400 RPM, and the induction phase extended out to 163 h.

    摘要翻译： 本发明涉及一种在对抗毒素抗性进行突变的巴斯德毕赤酵母菌株中表达免疫毒素的方法，包括：a）在含有蛋白质和酵母提取物的酶促消化物的生长培养基中培养巴斯德毕赤酵母，并将溶解氧浓度保持在40％以上 ; 和b）在诱导期间以0.5-0.75ml / min / IOL的初始体积的有限甲醇进料进行甲醇诱导，同时在低于17.5℃的温度下连续输注酵母提取物，消泡剂提供高达0.07％ ，搅拌降至400RPM，诱导期延长至163小时。

    Methods for expression and purification of immunotoxins
    8.
    发明申请
    Methods for expression and purification of immunotoxins 有权
    免疫毒素的表达和纯化方法

    公开(公告)号：US20060216782A1

    公开(公告)日：2006-09-28

    申请号：US10566886

    申请日：2004-08-02

    申请人： David Neville ,  Jung-Hee Woo ,  Yuan-Yi Liu

    发明人： David Neville ,  Jung-Hee Woo ,  Yuan-Yi Liu

    IPC分类号： C12P21/06 ,  C07H21/04 ,  C12N15/74 ,  C12N1/18 ,  C07K14/195

    CPC分类号： C07K1/18 ,  A61K47/6829 ,  A61K47/6849 ,  C07K16/2809 ,  C07K2317/622 ,  C07K2319/00 ,  C12N1/16

    摘要： In one aspect the present invention relates to a method of expressing an immunotoxin in Pichia pastoris strain mutated to toxin resistance comprising a) growing the Pichia pastoris in a growth medium comprising an enzymatic digest of protein and yeast extract and maintaing a dissolved oxygen concentration at 40% and above; and b) performing methanol induction with a limited methanol feed of 0.5-0.75 ml/min/10 L of initial volume during induction along with a continuous inusion of yeast extract at a temperature below 17.5° C., antifoaming agent supplied up to 0.07%, agitation reduced to 400 RPM, and the induction phase extended out to 163 h. In another aspect, the present invention relates to a method of purifying a nonglycosylated immunotoxin comprising a) loading a solution containing the nonglycosylated immunotoxin onto a hydrophobic interaction column; b) obtaining a first non-glycosylated immunotoxin containing eluant from the hydrophobic interaction column; c) loading the non-glycosylated immunotoxin containing eluant from step (b) onto an anion exchange column; d) obtaining a second non-glycosylated immunotoxin containing eluant from the anion exchange column by eluting the non-glycosylated immunotoxin with a sodium borate solution; e) diluting the concentration of sodium borate in the second non-glycosylated immunotoxin containing eluant from step (d) to about 50 mM or less; f) concentrating the diluted non-glycosylated immunotoxin containing eluant from step (e) over an anion exchange column; and g) obtaining a purified non-glycosylated immunotoxin from the anion exchange column.

    摘要翻译： 一方面，本发明涉及在对抗毒素抗性进行突变的巴斯德毕赤酵母菌株中表达免疫毒素的方法，其包括：a）在包含蛋白质和酵母提取物的酶促消化物的生长培养基中培养巴斯德毕赤酵母，并在40℃维持溶解氧浓度 ％ 以上; 和b）在诱导期间以0.5-0.75ml / min / 10L初始体积的有限甲醇进料进行甲醇诱导，同时在低于17.5℃的温度下连续引入酵母提取物，提供高达0.07％的消泡剂， ，搅拌降至400RPM，诱导期延长至163小时。 另一方面，本发明涉及纯化非糖基化免疫毒素的方法，其包括：a）将含有非糖基化免疫毒素的溶液装载到疏水相互作用柱上; b）从疏水相互作用柱获得含有第一非糖基化免疫毒素的洗脱液; c）将来自步骤（b）的含有非糖基化免疫毒素的洗脱液加载到阴离子交换柱上; d）通过用硼酸钠溶液洗脱非糖基化免疫毒素从阴离子交换柱获得含有第二非糖基化免疫毒素的洗脱液; e）将来自步骤（d）的第二非糖基化免疫毒素洗脱液中的硼酸钠浓度稀释至约50mM或更低; f）将来自步骤（e）的稀释的非糖基化的含有免疫毒素的洗脱液浓缩在阴离子交换柱上; 和g）从阴离子交换柱获得纯化的非糖基化免疫毒素。

    Methods for expression and purification of immunotoxins
    9.
    发明授权
    Methods for expression and purification of immunotoxins 有权
    免疫毒素的表达和纯化方法

    公开(公告)号：US08921097B2

    公开(公告)日：2014-12-30

    申请号：US12957165

    申请日：2010-11-30

    申请人： David M. Neville, Jr. ,  Jung-Hee Woo ,  Yuan-Yi Liu

    发明人： David M. Neville, Jr. ,  Jung-Hee Woo ,  Yuan-Yi Liu

    IPC分类号： C07K1/00 ,  C07K16/46 ,  C07K1/18 ,  C07K1/20 ,  C07K1/22 ,  C12N1/19

    CPC分类号： C07K1/18 ,  A61K47/6829 ,  A61K47/6849 ,  C07K16/2809 ,  C07K2317/622 ,  C07K2319/00 ,  C12N1/16

    摘要： The present invention relates to a method of expressing an immunotoxin in Pichia pastoris strain mutated to toxin resistance comprising a) growing the Pichia pastoris in a growth medium comprising an enzymatic digest of protein and yeast extract and maintaining a dissolved oxygen concentration at 40% and above; and b) performing methanol induction with a limited methanol feed of 0.5-0.75 ml/min/IO L of initial volume during induction along with a continuous infusion of yeast extract at a temperature below 17.5° C., antifoaming agent supplied up to 0.07%, agitation reduced to 400 RPM, and the induction phase extended out to 163 h.

    摘要翻译： 本发明涉及一种在对抗毒素抗性进行突变的巴斯德毕赤酵母菌株中表达免疫毒素的方法，包括：a）在含有蛋白质和酵母提取物的酶促消化物的生长培养基中培养巴斯德毕赤酵母，并将溶解氧浓度保持在40％以上 ; 和b）在诱导期间以0.5-0.75ml / min / IOL的初始体积的有限甲醇进料进行甲醇诱导，同时在低于17.5℃的温度下连续输注酵母提取物，消泡剂提供高达0.07％ ，搅拌降至400RPM，诱导期延长至163小时。