PROTEINS CARRYING ONE OR MORE UNNATURAL AMINO ACIDS

携带一种或多种非天然氨基酸的蛋白质

• 批准号: 6731307
• 负责人: UTTAM L RAJBHANDARY
• 金额: $ 28.86万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731307
• 关键词: acylation; aminoacid; aminoacid analog; aminoacid tRNA ligase; high performance liquid chromatography; method development; mutant; nucleic acid chemical synthesis; nucleic acid purification; nucleic acid sequence; peptide chemical synthesis; polymerase chain reaction; protein purification; protein structure function; transfer RNA; transposon /insertion element

DESCRIPTION (provided by applicant): The major objective of this proposal is to develop methods for the site specific insertion of one or more amino acids analogues into proteins in eubacteria and in eukaryotes. The amino acid analogues to be used could include those that are photoactivatable, those that are fluorescent, those that carry reactive side chains such as keto and azido groups, heavy atoms such as iodine, spectroscopic probes and those that mimic phospho-amino acids. Besides providing a method for the production of proteins with novel chemical and biological properties, proteins carrying such amino acid analogues will have wide applications in biology including studies on the folding, structure, stability and function of proteins, protein-protein interactions, protein localization and protein dynamics in vivo, crystallographic structure analyses and signal transduction. Two approaches are proposed. The first consists of the use of an amber suppressor transfer RNA (tRNA) aminoacylated in vivo with an amino acid analogue with the help of a mutant aminoacyl-tRNA synthetase, to insert the amino acid analogue at a specific site in a protein. A second, more general, approach is based on the finding in the investigator's laboratory that suppressor tRNAs, including those that are aminoacylated, can be imported into mammalian cells. This finding opens up the possibility of importing suppressor tRNAs aminoacylated in vitro with a variety of amino acid analogues for the site specific insertion of one or more amino acid analogues into proteins. The specific aims are: (1)To isolate mutants of (i) yeast tyrosyl-tRNA synthetase, which aminoacylate amber suppressor tRNA with iodotyrosine, p-trifluoromethyl-diazirinylphenylalanine or p-azido phenylalanine and (ii) E. coli glutaminyl-tRNA synthetase, which aminoacylate amber suppressor tRNA with 2-amino 5-keto hexanoic acid, an analogue of glutamine in which the side chain -CH2CH2CONH2 is replaced by - CH2CH2COCH3. (2) To identify, produce and purify amber and ochre suppressor tRNAs, which are not aminoacylated by mammalian aminoacyl-tRNA synthetases and which are highly active in suppression in mammalian cells. (3) To optimize the conditions for import of suppressor tRNAs aminoacylated with amino acid analogues into mammalian cells and to use them for site specific insertion into proteins.

描述（由申请人提供）： 该提案的主要目的是开发用于将一个或多个氨基酸类似物特异性插入到生态和真核生物中的蛋白质的方法。要使用的氨基酸类似物可以包括光活化的氨基酸类似物，荧光的氨基酸类似物，那些携带反应性侧链的氨基酸类似物，例如酮和氮杂基团，碘原子，例如碘，光谱探针以及模拟磷酸氨基酸的含量。除了提供一种具有新型化学和生物学特性的蛋白质的方法外，携带这种氨基酸类似物的蛋白质还将在生物学中具有广泛的应用，包括有关蛋白质折叠，结构，稳定性和功能的研究，蛋白质 - 蛋白质相互作用，蛋白质相互作用，蛋白质局部化和蛋白质的位置和蛋白质动力学在VIV，晶体结构和信号传输中。 提出了两种方法。第一个由使用氨基酸类似物的琥珀抑制器转移RNA（TRNA）氨基酰基氨基酰基组成，借助突变氨基酰基-TRNA合成酶在蛋白质中的特定位点插入氨基酸类似物。第二种，更通用的方法是基于研究人员实验室中的发现，即抑制TRNA（包括氨基酰胺的TRNA）可以进口到哺乳动物细胞中。这一发现打开了在体外与各种氨基酸类似物在体外进口抑制剂TRNA氨基酰基的可能性，以特异性插入一个或多个氨基酸类似物中。 The specific aims are: (1)To isolate mutants of (i) yeast tyrosyl-tRNA synthetase, which aminoacylate amber suppressor tRNA with iodotyrosine, p-trifluoromethyl-diazirinylphenylalanine or p-azido phenylalanine and (ii) E. coli glutaminyl-tRNA synthetase, which aminoacylate amber suppressor tRNA与2-氨基5-酮己酸六烯酸，谷氨酰胺的类似物侧链-CH2CH2CONH2被-CH2CH2COCH3取代。 （2）识别，生产和净化琥珀和OCHER抑制剂TRNA，这些TRNA并非由哺乳动物氨基酰基-TRNA合成酶氨基酰基化，并且在哺乳动物细胞中抑制高度活跃。 （3）优化用氨基酸类似物进口抑制剂TRNA氨基酰基的条件，并将其用于现场特异性插入蛋白质。