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1.发明授权PRODUCTION OF RECOMBINANT PROTEINS BY AUTOPROTEOLYTIC CLEAVAGE OF A FUSION PROTEIN 有权重组蛋白通过融合蛋白的自我蛋白酶解
公开(公告)号：EP1874932B1
公开(公告)日：2012-10-17
申请号：EP06721223.3
申请日：2006-04-25
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公开(公告)号：EP2935577B1
公开(公告)日：2017-10-04
申请号：EP13817679.7
申请日：2013-12-19
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3.发明公开METHOD FOR PRODUCING A RECOMBINANT PROTEIN OF INTEREST 有权VERFAHREN ZUR HERSTELLUNG EINES REKOMBINANTEN PROTEINS VON INTERESSE
公开(公告)号：EP2935577A1
公开(公告)日：2015-10-28
申请号：EP13817679.7
申请日：2013-12-19
摘要： Disclosed is a method for producing a recombinant protein of interest which is characterised by the following steps: (a) providing a first part of an N
pro autoprotease and providing a second part of an N
pro autoprotease, wherein said second part is fused by a peptidic bond to a protein of interest but said second part alone does not exhibit a proteolytic activity, and wherein complementation of said first part with the second part forms an autoproteolytically active N
pro autoprotease, (b) contacting the first part of the N
pro autoprotease with the second part of the N
pro autoprotease so that an autoproteolytically active N
pro autoprotease is formed and the protein of interest fused by the peptidic bond to the second part of the N
pro autoprotease is proteolytically cleaved off the second part of the N
pro autoprotease at the peptidic bond, and (c) recovering the protein of interest.摘要翻译： 本发明公开了一种生产重组蛋白质的方法，其特征在于以下步骤：（a）提供第一部分N亲自蛋白酶，并提供第二部分N亲自蛋白酶，其中所述第二部分通过 与目的蛋白质的肽键单独但仅表示第二部分不表现出蛋白水解活性，并且其中所述第一部分与第二部分的互补形成自动蛋白水解活性N亲自蛋白酶，（b）使第一部分N亲自蛋白酶 与N亲自蛋白酶的第二部分形成自由基蛋白水解活性的N亲自蛋白酶，并且通过肽键与N亲代自分泌蛋白酶的第二部分融合的目标蛋白质被蛋白水解地切割出N亲自蛋白酶的第二部分 在肽键处，和（c）回收目的蛋白质。
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公开(公告)号：EP2935576B1
公开(公告)日：2018-10-31
申请号：EP13811532.4
申请日：2013-12-19
CPC分类号： C12P21/06 , C07K2319/50 , C12N9/506 , C12N15/70 , C12P21/02
摘要： Disclosed is a method for producing a recombinant protein of interest, the method being characterised in by the following steps: (a) providing a fusion protein comprising an N pro autoprotease moiety and a protein of interest moiety in inclusion bodies, (b) solubilising the inclusion bodies, (c) allowing the fusion protein to be cleaved by the N pro autoprotease moiety under chaotropic conditions, wherein the recombinant protein of interest is cleaved from the fusion protein and wherein the recombinant protein of interest is not yet renatured or simultaneously renatured, and (d) recovering the protein of interest, optionally including a renaturing step for the protein of interest.
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公开(公告)号：EP2935576A1
公开(公告)日：2015-10-28
申请号：EP13811532.4
申请日：2013-12-19
CPC分类号： C12P21/06 , C07K2319/50 , C12N9/506 , C12N15/70 , C12P21/02
摘要： Disclosed is a method for producing a recombinant protein of interest, the method being characterised in by the following steps: (a) providing a fusion protein comprising an N pro autoprotease moiety and a protein of interest moiety in inclusion bodies, (b) solubilising the inclusion bodies, (c) allowing the fusion protein to be cleaved by the N pro autoprotease moiety under chaotropic conditions, wherein the recombinant protein of interest is cleaved from the fusion protein and wherein the recombinant protein of interest is not yet renatured or simultaneously renatured, and (d) recovering the protein of interest, optionally including a renaturing step for the protein of interest.
摘要翻译： 公开了一种产生目的重组蛋白的方法，所述方法的特征在于以下步骤：（a）提供包含N pro自体蛋白酶部分和目的蛋白质部分的融合蛋白在包涵体中，（b）将所述融合蛋白溶解 （c）允许所述融合蛋白在离液条件下被所述N pro自体蛋白酶部分切割，其中所述重组目的蛋白从所述融合蛋白切割并且其中所述重组蛋白尚未复性或同时复性， 和（d）回收感兴趣的蛋白质，任选地包括感兴趣的蛋白质的复性步骤。
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公开(公告)号：EP1874825B1
公开(公告)日：2010-03-24
申请号：EP06721225.8
申请日：2006-04-25
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7.发明公开PRODUCTION OF RECOMBINANT PROTEINS BY AUTOPROTEOLYTIC CLEAVAGE OF A FUSION PROTEIN 有权通过融合蛋白的自动切割生产重组蛋白质
公开(公告)号：EP1874932A2
公开(公告)日：2008-01-09
申请号：EP06721223.3
申请日：2006-04-25
摘要： The invention relates to a process for the recombinant production of a heterologous polypeptide of interest, comprising, (i) cultivation of a bacterial host cell which is transformed with an expression vector which comprises a nucleic acid molecule which codes for a fusion polypeptide, the fusion polypeptide comprising a derivative of an autoprotease Npro of Pestivirus, wherein at least one cysteine residue of the naturally occuring autoprotease Npro of Pestivirus is replaced by another amino acid residue, and a second polypeptide which is connected to the first polypeptide at the C-terminus of the first polypeptide in a manner such, that the second polypeptide is capable of being cleaved from the fusion polypeptide by the autoproteolytic activity of the first polypeptide, said second polypeptide being a heterologous polypeptide, wherein cultivation occurs under conditions which cause expression of the fusion polypeptide and formation of corresponding cytoplasmic inclusion bodies, (ii) isolation of the inclusion bodies from the host cell, (iii) solubilization of the isolated inclusion bodies, (iv) induction of autoproteolytic cleavage of the heterologous polypeptide of interest from the fusion polypeptide, and (v) isolation of the cleaved heterologous polypeptide of interest.
摘要翻译： 本发明涉及用于重组生产异源目的多肽的方法，其包括（i）培养用表达载体转化的细菌宿主细胞，所述表达载体包含编码融合多肽的核酸分子，融合物 包含瘟病毒自体蛋白酶Npro的衍生物的多肽，其中瘟病毒的天然存在的自噬蛋白酶Npro的至少一个半胱氨酸残基被另一个氨基酸残基置换，以及第二多肽，其在C末端与第一多肽连接 所述第一多肽以这样的方式存在，即所述第二多肽能够通过所述第一多肽的自体蛋白水解活性从所述融合多肽切割，所述第二多肽是异源多肽，其中培养在引起所述融合多肽表达的条件下发生 和形成相应的细胞质包涵体，（ii） （iii）分离的包涵体的溶解，（iv）诱导来自融合多肽的目的异源多肽的自体蛋白水解切割，和（v）分离目的切割的异源多肽 。
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公开(公告)号：EP1874825A2
公开(公告)日：2008-01-09
申请号：EP06721225.8
申请日：2006-04-25
发明人： JUNGBAUER, Alois , HAHN, Rainer , KAAR, Waltraud , SEIFERT, Michael , AUER, Bernhard , ACHMÜLLER, Clemens , WECHNER, Philipp
摘要： Disclosed is an affinity matrix comprising a solid phase and an affinity ligand comprising peptide bonds coupled to this solid phase, wherein the affinity ligand comprising peptide bond is selected from the following group of ligands: a) peptides comprising the formula X1X2X3X4, wherein X1 to X4 are amino acid residues and at least two of X1 to X4 is W, Y or F; b) peptides comprising the formula X5X6X7X8, wherein X5 to X8 are amino acid residues, at least one of X5 to X8 is W, and at least one of X5 to X8 is E or D; and c) poly-amino acids consisting of an amino acid monomer of the group consisting of R, K, E and D and an amino acid monomer of the group consisting of Y, F and W, preferably poly-KY, poly-KF, poly-KW, poly-RY, poly-RF, poly-RW, poly-EY, poly-DY, poly-EF, poly-EW, poly-DF and poly-DW, with the proviso that the peptides according to a) and b) have a maximum length of 35 amino acid residues and that the poly-amino acids according to c) have a minimum length of 20 amino acid residues.
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