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1. 发明申请

WO2015193897A1 GENETICALLY EXPANDED CELL FREE PROTEIN SYNTHESIS SYSTEMS, METHODS AND KITS 审中-公开
标题翻译：基因扩增无细胞免疫蛋白合成系统，方法和工具
公开(公告)号：WO2015193897A1
公开(公告)日：2015-12-23
申请号：PCT/IL2015/050616
申请日：2015-06-17
申请人： B. G. NEGEV TECHNOLOGIES AND APPLICATIONS LTD., AT BEN-GURION UNIVERSITY , ALFONTA, Lital , CHEMLA, Yonatan , OZER, Eden
发明人： B. G. NEGEV TECHNOLOGIES AND APPLICATIONS LTD., AT BEN-GURION UNIVERSITY , ALFONTA, Lital , CHEMLA, Yonatan , OZER, Eden
IPC分类号： C12P21/02 , C12P13/00
CPC分类号： C12P21/00 , C12N9/93 , C12P13/005 , C12P21/02 , C12Y601/01001 , C12Y601/01026
摘要： This invention relates to methods of producing a rare amino acid- or non-natural amino acid-containing protein in a cell free protein synthesis system and kits for use in and for accomplishing same. Specifically, the methods comprise the steps of expressing at least one orthogonal suppressor tRNA (o-tRNA)/aminoacyl-tRNA synthetase (aaRS) pair or derivatives thereof specific for incorporation of a rare amino acid- or non-natural amino acid in an E. coli organism; preparing a lysate of said E. coli organism expressing said orthogonal suppressor tRNA (o- tRNA)/aminoacyl-tRNA synthetase (aaRS) pair; and contacting said lysate with a template DNA containing a mutant gene in which at least one amino acid codon at a given site of the protein- encoding gene has been mutated into an amber or ochre mutation and further providing a cognate rare amino acid or non-natural amino acid and other factors necessary for protein synthesis; wherein protein synthesis occurs following said contact to produce a protein containing said at least one rare amino acid or said non-natural amino acid. Kits for use are described, as well.
摘要翻译：本发明涉及在无细胞蛋白质合成系统中生产稀有氨基酸或非天然氨基酸的蛋白质的方法以及用于和用于实现其的试剂盒。具体地说，该方法包括以下步骤：在E中特异性地掺入稀有氨基酸或非天然氨基酸的至少一种正交抑制基因tRNA（o-tRNA）/氨基酰-tRNA合成酶（aaRS）对或其衍生物大肠杆菌生物; 制备表达所述正交抑制子tRNA（o-tRNA）/氨酰-tRNA合成酶（aaRS）对的所述大肠杆菌生物的裂解物; 并使所述裂解物与含有突变基因的模板DNA接触，其中所述蛋白质编码基因的给定位点处的至少一个氨基酸密码子突变为琥珀色或赭色突变，并进一步提供相关的稀有氨基酸或非 - 天然氨基酸等蛋白质合成所必需的因素; 其中在所述接触之后发生蛋白质合成以产生含有所述至少一种稀有氨基酸或所述非天然氨基酸的蛋白质。也可以使用套件。

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