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1. 发明申请

WO2013149729A3 PROINSULIN WITH ENHANCED HELPER SEQUENCE 审中-公开
标题翻译： PROINSULIN具有增强的HELPER序列
公开(公告)号：WO2013149729A3
公开(公告)日：2013-12-05
申请号：PCT/EP2013001003
申请日：2013-04-04
申请人： GLUCOMETRIX AG
发明人： ECKERT KELVIN , HERMANN STEFAN
IPC分类号： C07K14/62
CPC分类号： C07K14/62 , C07K2319/00 , C07K2319/21
摘要： The invention provides a polypeptide comprising an insulin precursor and the N-terminal helper sequence MAKR. The helper sequence enhances the expression of the insulin precursor in a recombinant organism and increases the yields of insulin after purification and processing of the insulin precursor. The invention is thus further concerned with a method of producing insulin, in which proinsulin is expressed as recombinant polypeptide in an organism such as E. coli, isolated from the organism and then processed to yield active insulin.
摘要翻译：本发明提供了包含胰岛素前体和N-末端辅助序列MAKR的多肽。该辅助序列增强重组生物体中胰岛素前体的表达，并增加胰岛素前体纯化和加工后的胰岛素产量。因此，本发明还涉及生产胰岛素的方法，其中胰岛素原在生物体如大肠杆菌中作为重组多肽表达，从生物体中分离，然后加工产生活性胰岛素。

2. 发明申请

WO2012152439A1 COMPREHENSIVE BUFFER SYSTEM FOR RENATURING HUMAN PROINSULIN OR PROINSULIN DERIVATIVES 审中-公开
标题翻译：用于重新培养人胰蛋白酶或蛋白酶衍生物的综合缓冲系统
公开(公告)号：WO2012152439A1
公开(公告)日：2012-11-15
申请号：PCT/EP2012/001992
申请日：2012-05-09
申请人： GLUCOMETRIX AG , ECKERT, Kelvin , HERMANN, Stefan , NOACK, Janina , SCHWEDE, Matthias
发明人： ECKERT, Kelvin , HERMANN, Stefan , NOACK, Janina , SCHWEDE, Matthias
IPC分类号： C07K14/62
CPC分类号： C07K14/62
摘要： The invention relates to an improved processing and purification method for the production of proinsulin or derivatives thereof starting from the solubilisation of inclusion bodies isolated from a prokaryotic expression system, such as E. coli, whereby all processing (i.e. refolding, precipitation, concentration) and purification steps are carried out in the same aqueous buffer system comprising an organic amphoteric substance containing at least two functional groups, which contain at least an amino and a carboxylic acid group, and which is characterized by a difference in the pKa values of its at least one amino group and at least one carboxylic acid group of at least 7. Furthermore, the organic amphoteric substance is an efficient scavenger of cyanate, thereby preventing irreversible carbamylation of primary amines present in proinsulin or the derivates thereof, which has been observed in the presence of high concentrations of urea. Thus, the disadvantage associated with this widespread chaotropic auxiliary is obliterated, allowing the utilization of efficient and uncomplicated chromatography techniques for purification steps subsequent to solubilisation and refolding. The use of an efficient buffer substance in all processing steps from inclusion body solubilisation up to the re-folded, concentrated and purified precursor of insulin or a derivate thereof, which at the same time acts as an agent preventing protein modification by carbamylation, thereby abolishing the need to add an additional compound with scavenging activity, leads to significant advantages regarding the efficiency and efficacy of the procedure compared to previously employed production methods.
摘要翻译：本发明涉及从原核表达系统如大肠杆菌分离的包涵体的溶解开始，用于生产胰岛素原或其衍生物的改进的加工和纯化方法，其中所有加工（即重折叠，沉淀，浓缩）和纯化步骤在含有至少两个含有至少一个氨基和羧酸基团的官能团的有机两性物质的相同的水性缓冲体系中进行，其特征在于其至少其pKa值的差异一个氨基和至少一个至少一个羧酸基团至少为7.此外，有机两性物质是有效的氰酸盐清除剂，从而防止存在于胰岛素原中的伯胺或其衍生物的不可逆氨基甲酰化，这在存在下观察到的高浓度尿素。因此，与这种广泛的离液助剂相关的缺点被消除，允许利用有效和不复杂的色谱技术用于溶解和再折叠后的纯化步骤。在所有加工步骤中使用有效缓冲物质从包涵体溶解直至胰岛素的再折叠，浓缩和纯化前体或其衍生物，其同时用作通过氨基甲酰化抑制蛋白质修饰的试剂，从而消除与以前使用的生产方法相比，需要添加具有清除活性的另外的化合物，导致与该方法相比的效率和效力方面的显着优点。

3. 发明申请

WO2012104099A1 PROCESS FOR THE PRODUCTION OF RECOMBINANT TRYPSIN 审中-公开
标题翻译：生产重组性TRYPSIN的方法
公开(公告)号：WO2012104099A1
公开(公告)日：2012-08-09
申请号：PCT/EP2012/000497
申请日：2012-02-03
申请人： GLUCOMETRIX AG , KÖNIG, Peter
发明人： KÖNIG, Peter
IPC分类号： C12N9/76
CPC分类号： C12N9/6427 , C07K2319/21
摘要： The invention relates to a process for the production of recombinant trypsin and its use for processing of an insulin precursor into insulin. In particular, the invention relates to a process for the production of recombinant trypsin starting from recombinant trypsinogen produced in a prokaryotic host cell. In the process of the invention the recombinant trypsinogen is produced by the host cell in form of inclusion bodies. The trypsinogen contained in the inclusion bodies is purified and subsequently refolded into its native conformation involving formation of disulfide bridges. The refolded trypsinogen is then further processed into active trypsin.
摘要翻译：本发明涉及生产重组胰蛋白酶的方法及其用于将胰岛素前体加工成胰岛素的用途。特别地，本发明涉及一种从原核宿主细胞中产生的重组胰蛋白酶原产生重组胰蛋白酶的方法。在本发明的方法中，重组胰蛋白酶原由宿主细胞以包涵体的形式产生。包含在包涵体中的胰蛋白酶原被纯化并随后重折叠成其涉及形成二硫键的天然构象。然后将重折叠的胰蛋白酶原进一步加工成活性胰蛋白酶。

4. 发明申请

WO2013149729A2 PROINSULIN WITH ENHANCED HELPER SEQUENCE 审中-公开
标题翻译： PROINSULIN具有增强的帮助序列
公开(公告)号：WO2013149729A2
公开(公告)日：2013-10-10
申请号：PCT/EP2013/001003
申请日：2013-04-04
申请人： GLUCOMETRIX AG
发明人： ECKERT, Kelvin , HERMANN, Stefan
IPC分类号： C07K14/435
CPC分类号： C07K14/62 , C07K2319/00 , C07K2319/21
摘要： The invention provides a polypeptide comprising an insulin precursor and the N-terminal helper sequence MAKR. The helper sequence enhances the expression of the insulin precursor in a recombinant organism and increases the yields of insulin after purification and processing of the insulin precursor. The invention is thus further concerned with a method of producing insulin, in which proinsulin is expressed as recombinant polypeptide in an organism such as E. coli, isolated from the organism and then processed to yield active insulin.
摘要翻译：本发明提供了包含胰岛素前体和N-末端辅助子序列MAKR的多肽。辅助序列增强胰岛素前体在重组生物体中的表达并增加胰岛素前体纯化和加工后的胰岛素产量。因此，本发明进一步涉及一种生产胰岛素的方法，其中胰岛素原在从生物体分离的生物体如大肠杆菌中表达为重组多肽，然后加工产生活性胰岛素。

5. 发明申请

WO2012098009A1 CHIMERIC POLYPEPTIDE COMPRISING A MEMBRANE PROTEIN AND AN INSULIN PRECURSOR 审中-公开
标题翻译：包含膜蛋白和胰岛素前体的CHIMERIC POLYPEPTIS
公开(公告)号：WO2012098009A1
公开(公告)日：2012-07-26
申请号：PCT/EP2012/000266
申请日：2012-01-20
申请人： GLUCOMETRIX AG , PONOMARENKO, Sophia
发明人： PONOMARENKO, Sophia
IPC分类号： C07K14/62 , A61K38/28 , C12N9/14
CPC分类号： C07K14/62 , A61K38/00 , C07K2319/00 , C07K2319/03 , C07K2319/21 , C07K2319/50 , C12N9/14
摘要： The present invention provides a chimeric polypeptide and an isolated nucleic acid such as DNA coding for the chimeric polypeptide comprising i) a first peptidyl fragment, comprising an amino acid sequence of the ATP synthase subunit F0C, and ii) a second peptidyl fragment which is an insulin precursor. In a preferred embodiment, the insulin precursor comprises insulin chains A and B, and most preferably is human proinsulin. The invention further provides a method of producing a recombinant chimeric polypeptide and a method of producing insulin. In the process of the invention disulfide bridges are formed in the recombinant polypeptide in vivo upon expression in the recombinant cell. The recombinant polypeptide can be obtained from the recombinant cell in semifolded form.
摘要翻译：本发明提供嵌合多肽和分离的核酸，例如编码嵌合多肽的DNA，其包含i）第一肽基片段，其包含ATP合酶亚基F0C的氨基酸序列，和ii）第二肽基片段，其为胰岛素前体。在优选的实施方案中，胰岛素前体包含胰岛素链A和B，最优选人胰岛素原。本发明还提供了生产重组嵌合多肽的方法和一种产生胰岛素的方法。在本发明的方法中，在重组细胞中表达后，体内重组多肽形成二硫键。可以从重组细胞以半成形形式获得重组多肽。

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