A new functional suppressor tRNA/aminoacyl-tRNA synthetase pair for the in vivo incorporation of unnatural amino acids into proteins
A new functional suppressor tRNA/aminoacyl-tRNA synthetase pair for the in vivo incorporation of unnatural amino acids into proteins
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DOI：
10.1021/ja000595y
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发表时间：
2000-05-24
影响因子：
15
通讯作者：
Schultz, PG
中科院分区：
文献类型：
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作者：
Wang, L;Magliery, TJ;Schultz, PG
直接从生长培养基中选择性地将非天然氨基酸掺入体内蛋白质中的一般方法将极大地扩展我们操纵蛋白质结构和功能的能力。例如，将荧光团选择性地置于体内蛋白质中的能力将为细胞生物学提供强大的工具，或者产生大量具有金属结合或酮基氨基酸的蛋白质的能力可能导致具有增强的物理或催化性质的蛋白质。我们的方法涉及产生一个抑制tRNA/氨酰-tRNA合成酶（tRNACUA/阿尔斯）对，是正交的大肠杆菌内源性tRNA/合成酶对，也就是说，正交的tRNA不是任何内源性合成酶的底物和正交合成酶不识别任何内源性tRNA。2，3然后改变该合成酶的特异性，使得其仅用所需的非天然氨基酸装载tRNACUA。一个这样的正交对用于E.大肠杆菌是从来自酿酒酵母的tRNA 2 Gln/GlnRS对发展而来的。另外的正交tRNA/阿尔斯对的开发可能允许多个非天然氨基酸同时掺入蛋白质中。此外，不同的氨酰合成酶可能是产生具有特定特异性的活性位点的更好的起点（例如，对大的疏水性氨基酸与小的亲水性氨基酸的特异性）。为此，我们分析了生物化学数据可用于tRNATyr/TyrRS对从各种生物。该分析与体内互补测定一起提供了新的正交tRNACUA Tyr/TyrRS对以及对其他对的开发的见解。原核tRNATyr的身份元件包括与真核tRNATyr的短臂相反的长可变臂。4此外，真核生物tRNATyr含有C1：G72正识别元件，而原核生物tRNATyr没有这样的共有碱基对。5，6体外研究也表明，S. cerevisiae 7和Homo sapiens 8不能被细菌合成酶氨酰化，它们的TyrRS也不能氨酰化细菌tRNA。为了检验这些微生物的tRNACUA Tyr/TyrRS对在E.在大肠杆菌中，使用基于抑制质粒pBLAM中编码的TEM-1 β-内酰胺酶基因的非必需位置中的琥珀终止密码子的体内互补测定。3如果新引入的抑制基因tRNACUA被任何内源性E.大肠杆菌合成酶，细胞将在氨苄青霉素存在下生长。在E.用pBLAM转化的大肠杆菌菌株DH 10 B，细胞在非常高浓度的氨苄青霉素下存活，对于来自S. cereVisiae和H.智人当S. cereVisiae tRNACUA Gln是一种正交tRNA，在相同条件下测试，细胞仅在20 μg/mL氨苄青霉素下存活。3作为比较，E.单独携带pBLAM的大肠杆菌菌株在高达9.7 μg/mL氨苄青霉素（在不存在任何抑制tRNA的情况下）下存活。由于合成酶对tRNA的识别取决于细胞中的相对浓度，因此通过在较弱的lac启动子而不是强的lpp启动子下表达其基因来降低tRNACUA Tyr的浓度。氨苄青霉素对S. cereVisiae和H. sapiens，但这些值仍然可能太高，不允许这些tRNA以正交对的形式使用。cereVisiae and H.
General methods for selectively incorporating unnatural amino acids into proteins in vivo, directly from the growth media, would greatly expand our ability to manipulate protein structure and function. 1 For example, the ability to place fluorophores selectively into proteins in vivo would provide powerful tools for cell biology, or the ability to generate large quantities of proteins with metal binding or keto amino acids might lead to proteins with enhanced physical or catalytic properties. Our approach involves the generation of a suppressor tRNA/aminoacyl-tRNA synthetase (tRNACUA/aaRS) pair that is orthogonal to Escherichia coli endogenous tRNA/synthetase pairs; that is, the orthogonal tRNA is not a substrate for any endogenous synthetases and the orthogonal synthetase does not recognize any endogenous tRNAs. 2, 3 The specificity of this synthetase is then altered so that it charges the tRNACUA only with a desired unnatural amino acid. One such orthogonal pair for use in E. coli was developed from the tRNA2 Gln/GlnRS pair from Saccharomyces cereVisiae. 3 The development of additional orthogonal tRNA/aaRS pairs may allow the simultaneous incorporation of multiple unnatural amino acids into proteins. Moreover, different aminoacyl synthetases may be better starting points for generating active sites with particular specificities (eg, specificity for large hydrophobic vs small hydrophilic amino acids). To this end, we have analyzed biochemical data available for tRNATyr/TyrRS pairs from a variety of organisms. This analysis, together with in vivo complementation assays, has afforded a new orthogonal tRNACUA Tyr/TyrRS pair as well as insights into the development of additional pairs. The identity elements of prokaryotic tRNATyr include a long variable arm in contrast to the short arm of eukaryotic tRNATyr. 4 In addition, eukaryotic tRNATyr contains a C1: G72 positive recognition element, whereas prokaryotic tRNATyr has no such consensus base pair. 5, 6 In vitro studies have also shown that tRNATyr of S. cereVisiae7 and Homo sapiens8 cannot be aminoacylated by bacterial synthetases, nor do their TyrRS aminoacylate bacterial tRNA. To test whether tRNACUA Tyr/TyrRS pairs from these organisms are orthogonal in E. coli, an in vivo complementation assay was used that is based on suppression of an amber stop codon in a nonessential position of the TEM-1 β-lactmase gene encoded in plasmid pBLAM. 3 If the newly introduced suppressor tRNACUA is aminoacylated by any endogenous E. coli synthetases, cells will grow in the presence of ampicillin. After expressing these tRNACUA Tyr in E. coli strain DH10B transformed with pBLAM, cells survive at very high concentrations of ampicillin, greater than 1206 μg/mL (interpolated from IC50 curves in Figure 1) for tRNACUA Tyr derived from S. cereVisiae and 234 μg/mL for that from H. sapiens. When S. cereVisiae tRNACUA Gln, which is an orthogonal tRNA, is tested under the same conditions, the cells survive at only 20 μg/mL ampicillin. 3 For comparison, E. coli strains bearing pBLAM alone survive up to 9.7 μg/mL ampicillin (in the absence of any suppressor tRNA). Since the recognition of tRNA by synthetase depends on relative concentrations in the cell, 9, 10 the concentration of tRNACUA Tyr was decreased by expressing its gene under the weaker lac promoter instead of the strong lpp promoter. The IC50’s decreased to 383 and 84 μg/mL ampicillin for S. cereVisiae and H. sapiens, respectively, but these values are still potentially too high to allow the use of these tRNAs in orthogonal pairs.The change of one single nucleotide in the anticodon (G34 to C34) made the S. cereVisiae and H …