公开(公告)号：US08298789B2

公开(公告)日：2012-10-30

申请号：US12452919

申请日：2007-12-06

申请人： Uma Sankararaman ,  Dipanwita Maiti ,  Meera Sankarankutty ,  Rakesh Shekhawat ,  Narasimha Kumar Kopparapu ,  Gulnaz Zaidi ,  Bipinchandra Rathod ,  Niren Praful Thakar ,  Priti Thakur ,  Anjali Chutke ,  Shrikant Mishra

发明人： Uma Sankararaman ,  Dipanwita Maiti ,  Meera Sankarankutty ,  Rakesh Shekhawat ,  Narasimha Kumar Kopparapu ,  Gulnaz Zaidi ,  Bipinchandra Rathod ,  Niren Praful Thakar ,  Priti Thakur ,  Anjali Chutke ,  Shrikant Mishra

IPC分类号： C12N15/16

CPC分类号： C07K14/635 ,  C07K2319/21 ,  C07K2319/35 ,  C07K2319/50 ,  C07K2319/61

摘要： The present invention discloses a process for the preparation of rhPTH (1-34) also known as teriparatide by construction of a novel nucleotide, as an NcoI.IXhoI fragment as set forth in SEQ. ID. No.:1 encoding a chimeric fusion protein as set forth in SEQ.ID. No.:2 comprising of a fusion partner consisting of 41 amino acids belonging to Escherichia coli β-galactosidase (LacZ) gene, an endopeptidase cleavage site, rhPTH (1-34) gene fragment, cloning the said nucleotide in an expression vector under the control of T7 promoter, transforming Escherichia coli with the said vector and expressing the chimeric fusion protein in fed batch fermentation. The present invention further discloses a low feed rate lactose induction for optimized expression of rhPTH (1-34) in Escherichia coli. The present invention also discloses an unique, novel two step orthogonal purification process for rhPTH (1-34) comprising of cation exchange chromatography optionally followed by preparative chromatography selected from HIC or RP-HPLC to yield a target protein of ≧99% purity. The present invention discloses a simple, cost-effective, environmentally benign method of producing high purity rhPTH (1-34).

摘要翻译： 本发明公开了一种制备rhPTH（1-34）的方法，其也称为特立帕肽，通过构建新的核苷酸，如SEQ ID NO：1所示的NcoI.IXhoI片段。 ID。 编号如SEQ.ID所示的嵌合融合蛋白。 编号：2包含由属于大肠杆菌和半乳糖苷酶（LacZ）基因的41个氨基酸组成的融合配偶体，内肽酶切割位点，rhPTH（1-34）基因片段，将所述核苷酸克隆在表达载体中 T7启动子的控制，用所述载体转化大肠杆菌并在补料分批发酵中表达嵌合融合蛋白。 本发明还公开了用于在大肠杆菌中优化rhPTH（1-34）表达的低进料速率乳糖诱导。 本发明还公开了一种用于rhPTH（1-34）的独特的新型两步正交纯化方法，其包括阳离子交换色谱，任选地随后进行选自HIC或RP-HPLC的制备型色谱，以产生≥99％纯度的靶蛋白。 本发明公开了一种生产高纯度rhPTH（1-34）的简单，成本效益高，环境友好的方法。

公开(公告)号：US20100145033A1

公开(公告)日：2010-06-10

申请号：US12452919

申请日：2007-12-06

申请人： Uma Sankararaman ,  Dipanwita Maiti ,  Meera Sankarankutty ,  Rakesh Shekhawat ,  Narasimha Kumar Kopparapu ,  Gulnaz Zaidi ,  Bipinchandra Rathod ,  Niren Praful Thakar ,  Priti Thakur ,  Anjali Chutke ,  Shrikant Mishra

发明人： Uma Sankararaman ,  Dipanwita Maiti ,  Meera Sankarankutty ,  Rakesh Shekhawat ,  Narasimha Kumar Kopparapu ,  Gulnaz Zaidi ,  Bipinchandra Rathod ,  Niren Praful Thakar ,  Priti Thakur ,  Anjali Chutke ,  Shrikant Mishra

IPC分类号： C07K14/635

CPC分类号： C07K14/635 ,  C07K2319/21 ,  C07K2319/35 ,  C07K2319/50 ,  C07K2319/61

摘要： The present invention discloses a process for the preparation of rhPTH (1-34) also known as teriparatide by con-struction of a novel nucleotide, as an NcoI.IXhoI fragemt as set forth in SEQ. ID. No.:1 encoding a chimeric fusion protein as set forth in SEQ.ID. No.:2 comprising of a fusion partner consisting of 41 amino acids belonging to Escherichia coli β-galactosidase (LacZ) gene, an endopeptidase cleavage site, rhPTH (1-34) gene fragment, cloning the said nucleotide in an expression vector under the control of T7 promoter, transforming Escherichia coli with the said vector and expressing the chimeric fusion protein in fed batch fermentation. The present invention further discloses a low feed rate lactose induction for optimized expression of rhPTH (1-34) in Escherichia coli. The present invention also discloses an unique, novel two step orthogonal purification process for rhPTH (1-34) comprising of cation exchange chromatography optionally followed by preparative chromatography selected from HIC or RP-HPLC to yield a target protein of ≧99% purity. The present invention discloses a simple, cost-effective, environmentally benign method of producing high purity rhPTH (1-34).

摘要翻译： 本发明公开了一种制备rhPTH（1-34）的方法，其也称为特立帕肽，通过构建新核苷酸作为SEQ ID NO：1所示的NcoI.IXhoI fragemt。 ID。 编号如SEQ.ID所示的嵌合融合蛋白。 编号：2包含由属于大肠杆菌和半乳糖苷酶（LacZ）基因的41个氨基酸组成的融合配偶体，内肽酶切割位点，rhPTH（1-34）基因片段，将所述核苷酸克隆在表达载体中 T7启动子的控制，用所述载体转化大肠杆菌并在补料分批发酵中表达嵌合融合蛋白。 本发明还公开了用于在大肠杆菌中优化rhPTH（1-34）表达的低进料速率乳糖诱导。 本发明还公开了一种用于rhPTH（1-34）的独特的新型两步正交纯化方法，其包括阳离子交换色谱，任选地随后进行选自HIC或RP-HPLC的制备型色谱，以产生≥99％纯度的靶蛋白。 本发明公开了一种生产高纯度rhPTH（1-34）的简单，成本效益高，环境友好的方法。