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1.发明公开Process of production of a recombinant polypeptide by fusion with an autoprotease 有权生产方法von einem rekombinant多肽纯合酶融合蛋白酶自身蛋白酶。
公开(公告)号：EP2366719A2
公开(公告)日：2011-09-21
申请号：EP11167687.0
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 N pro of Pestivirus, wherein at least one cysteine residue of the naturally occuring autoprotease N pro 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： 本发明涉及重组产生感兴趣的异源多肽的方法，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，所述融合 其包含天蛾病毒自身蛋白酶N pro的至少一个半胱氨酸残基被另一个氨基酸残基替代，第二个多肽与第一多肽在C-末端相连， 第一多肽的第一个多肽的末端以第二多肽能够通过第一多肽的自身蛋白水解活性从融合多肽切割，所述第二多肽是异源多肽，其中培养发生在引起表达的条件下 融合多肽和形成相应的细胞质包涵体，（i i）从宿主细胞中分离包涵体，（iii）分离的包涵体的溶解，（iv）从融合多肽诱导感兴趣的异源多肽的自体蛋白水解切割，和（v）分离的切割的异源多肽 出于兴趣。
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2.发明授权Process of production of a recombinant polypeptide by fusion with an autoprotease 有权通过与自体蛋白酶融合产生重组多肽的过程
公开(公告)号：EP2366719B1
公开(公告)日：2018-02-21
申请号：EP11167687.0
申请日：2006-04-25
摘要： Disclosed is a method for the production of a heterologous polypeptide of interest with a homogenous N-terminus, using a fusion polypeptide comprising the polypeptide of interest and N-terminally thereto a polypeptide exhibiting autoproteolytic function, said method comprising the steps of a) binding of the fusion polypeptide in a soluble, autoproteolytically inactive form by an affinity chromatography system, b) refolding of the fusion polypeptide, thereby activating the autoproteolytic function of the fusion polypeptide and causing cleavage of the heterologous polypeptide of interest, and c) subsequently eluting the heterologous polypeptide of interest, wherein said steps are conducted on one affinity chromatography system, especially a ligand selected from the following group of ligands: a) peptides comprising the formula X 1 X 2 X 3 X 4 , wherein X 1 to X 4 are amino acid residues and at least two of X 1 to X 4 is W, Y or F; b) peptides comprising the formula X 5 X 6 X 7 X 8 , wherein X 5 to X 8 are amino acid residues, at least one of X 5 to X 8 is W, and at least one of X 5 to X 8 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 polyKY, 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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3.发明公开Process of production of a recombinant polypeptide by fusion with an autoprotease 有权生产方法von einem rekombinant多肽纯合酶融合蛋白酶自身蛋白酶。
公开(公告)号：EP2366719A3
公开(公告)日：2012-03-07
申请号：EP11167687.0
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 N pro of Pestivirus, wherein at least one cysteine residue of the naturally occuring autoprotease N pro 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： 本发明公开了一种使用包含感兴趣多肽的融合多肽和其N-末端具有表达自身蛋白水解功能的多肽的同源N-末端产生感兴趣的异源多肽的方法，所述方法包括以下步骤：a） 通过亲和层析系统以可溶性，自旋蛋白水解失活形式的融合多肽，b）融合多肽的重折叠，由此激活融合多肽的自体蛋白水解功能并引起感兴趣的异源多肽的切割，以及c）随后将异源 多肽，其中所述步骤在一个亲和层析系统，特别是选自下列配体组的配体中进行：a）包含式X 1 X 2 X 3 X 4的肽，其中X 1至X 4为氨基酸 残基，X 1〜X 4中的至少2个为W，Y或F; b）包含式X 5 X 6 X 7 X 8的肽，其中X 5至X 8为氨基酸残基，X 5至X 8中的至少一个为W，X 5至X 8中的至少一个为E 或D; 和c）由由R，K，E和D组成的组的氨基酸单体和由Y，F和W组成的组的氨基酸单体组成的聚氨基酸，优选为聚KY，聚-KF，聚 - KW，poly-RY，poly-RF，poly-RW，poly-EY，poly-DY，poly-EF，poly-EW，poly-DF和poly-DW，条件是根据a）和b ）具有35个氨基酸残基的最大长度，并且根据c）的多氨基酸具有20个氨基酸残基的最小长度。
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公开(公告)号：EP3037530B1
公开(公告)日：2017-02-01
申请号：EP14199893.0
申请日：2014-12-22
申请人： SANDOZ AG
IPC分类号： C12N15/09
CPC分类号： C12N15/67 , C12N15/1072 , C12N15/70 , C12N2015/8518 , C12Q1/6858 , C12Q1/6869
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公开(公告)号：EP3037530A1
公开(公告)日：2016-06-29
申请号：EP14199893.0
申请日：2014-12-22
申请人： SANDOZ AG
IPC分类号： C12N15/09
CPC分类号： C12N15/67 , C12N15/1072 , C12N15/70 , C12N2015/8518 , C12Q1/6858 , C12Q1/6869
摘要： Amino acid residue misincorporations are necessarily found in sequence variants at low concentrations in admixture with expressed polypeptides, resulting from one or more base mismatches within codons susceptible to amino acid residue misincorporation during transcription and/or translation. The invention provides a method of optimizing the coding sequences of a polynucleotide that encodes a polypeptide, wherein at least one codon is susceptible to amino acid residue misincorporation. The method of the invention can be used to reverse-engineer an unknown coding sequence, which encodes the same polypeptide, but differs in said at least one codon from the known coding sequence. The method can further be used to alter the immunogenic potential of an expressed polypeptide. Thus, the invention is useful in engineering optimized polynucleotides encoding polypeptides.
摘要翻译： 必须在低浓度的序列变体中以与表达的多肽混合的序列变体中发现氨基酸残基错配菌株，其由在转录和/或翻译期间对氨基酸残基错配进行敏感的密码子内的一个或多个碱基错配产生。 本发明提供了一种优化编码多肽的多核苷酸的编码序列的方法，其中至少一个密码子易受氨基酸残基错配。 本发明的方法可用于逆转编码相同多肽的未知编码序列，但在所述至少一个密码子与已知编码序列中不同。 该方法还可用于改变表达多肽的免疫原性潜力。 因此，本发明可用于工程化编码多肽的优化多核苷酸。
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