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1. 发明申请

WO2005100585A2 METHODS FOR OBTAINING DIRECTIONALLY TRUNCATED POLYPEPTIDES 审中-公开
标题翻译：用于获得方向截断的多糖的方法
公开(公告)号：WO2005100585A2
公开(公告)日：2005-10-27
申请号：PCT/US2005/010811
申请日：2005-03-30
申请人： EPICENTRE , FIANDT, Michael, J. , DAHL, Gary, A.
发明人： FIANDT, Michael, J. , DAHL, Gary, A.
IPC分类号： C12P21/06
CPC分类号： C12P21/06 , C12N15/102 , C12N15/1082 , C12Q1/6883 , C12Q2600/142 , C12Q2600/156 , C12Q2600/158
摘要： Methods, compositions and kits are disclosed for obtaining directionally truncated polypeptides by inserting a transposon. Preferably the transposon comprises a selectable marker and an ori, and optionally a promoter, a ribosome binding site and a translation start codon, into a target sequence in vitro or in vivo. Amplification products, varying in length depending on the transposon insertion site, are obtained using one primer that anneals to the target sequence and a second primer that anneals to the transposon. Amplification products are ligated to circular dsDNA, transformed into host cells, and individual colonies, each containing a directionally truncated clone of the target sequence, are obtained by plating on medium for which the selectable marker encodes resistance. Directionally truncated polypeptides encoded by the target sequence are obtained in vivo by inducing an RNAP in the host cells that uses the promoter or, in vitro by cell-free transcription and translation.
摘要翻译：公开了通过插入转座子获得定向截短的多肽的方法，组合物和试剂盒。优选地，转座子在体外或体内包含选择标记和ori，以及任选的启动子，核糖体结合位点和翻译起始密码子。使用与靶序列退火的一种引物和与转座子退火的第二引物获得长度不同的扩增产物，这取决于转座子插入位点。将扩增产物连接到环状dsDNA上，转化到宿主细胞中，每个含有靶序列的方向截短的克隆的单个克隆通过在可选择标记编码抗性的培养基上电镀获得。通过在使用该启动子的宿主细胞中诱导RNAP或体外通过无细胞转录和翻译在体内获得由靶序列编码的定向截短的多肽。

2. 发明申请

WO2005100585A3 METHODS FOR OBTAINING DIRECTIONALLY TRUNCATED POLYPEPTIDES 审中-公开
标题翻译：用于获得方向截断的多糖的方法
公开(公告)号：WO2005100585A3
公开(公告)日：2006-05-04
申请号：PCT/US2005010811
申请日：2005-03-30
申请人： EPICT , FIANDT MICHAEL J , DAHL GARY A
发明人： FIANDT MICHAEL J , DAHL GARY A
IPC分类号： C12P21/00 , C12P19/34 , C12P21/06 , C12Q1/68
CPC分类号： C12P21/06 , C12N15/102 , C12N15/1082 , C12Q1/6883 , C12Q2600/142 , C12Q2600/156 , C12Q2600/158
摘要： Methods, compositions and kits are disclosed for obtaining directionally truncated polypeptides by inserting a transposon. Preferably the transposon comprises a selectable marker and an ori, and optionally a promoter, a ribosome binding site and a translation start codon, into a target sequence in vitro orin vivo. Amplification products, varying in length depending on the transposon insertion site, are obtained using one primer that anneals to the target sequence and a second primer that anneals to the transposon. Amplification products are ligated to circular dsDNA, transformed into host cells, and individual colonies, each containing a directionally truncated clone of the target sequence, are obtained by plating on medium for which the selectable marker encodes resistance. Directionally truncated polypeptides encoded by the target sequence are obtained in vivo by inducing an RNAP in the host cells that uses the promoter or,in vitro by cell-free transcription and translation.
摘要翻译：公开了通过插入转座子获得定向截短的多肽的方法，组合物和试剂盒。优选地，转座子在体外或体内包含选择性标记和ori，以及任选的启动子，核糖体结合位点和翻译起始密码子。使用与靶序列退火的一种引物和与转座子退火的第二引物获得长度不同的扩增产物，这取决于转座子插入位点。将扩增产物连接到环状dsDNA上，转化到宿主细胞中，每个含有靶序列的方向截短的克隆的单个克隆通过在可选择标记编码抗性的培养基上电镀获得。通过在使用该启动子的宿主细胞中诱导RNAP或体外通过无细胞转录和翻译在体内获得由靶序列编码的定向截短的多肽。

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