Biosynthesis of Proteins Incorporating a Versatile Set of Phenylalanine Analogues.
Biosynthesis of Proteins Incorporating a Versatile Set of Phenylalanine Analogues.
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包含一组多功能苯丙氨酸类似物的蛋白质的生物合成。
DOI：
10.1002/chin.200222183
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
D. Tirrell
中科院分区：
文献类型：
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作者：
K. Kirshenbaum;I. Carrico;D. Tirrell
大分子化学面临着一个二分法。化学家可以制备具有多种官能团的聚合物，但无法获得蛋白质和核酸的序列特异性和单分散性。相反，蛋白质的化学多样性受到遗传密码指定的少量氨基酸的严重限制。我们能否找到方法将合成聚合物化学的多样性与蛋白质生物合成的精确性结合起来？一种方法是提高蛋白质生物合成装置利用二十种典型氨基酸以外的单体的能力。[1,2]特别关注的是氨基酰基trna合成酶（aaRS），它将氨基酸结合到它们的同源trna上。tRNA充电的特异性对于确保遗传信息翻译成蛋白质序列的保真度至关重要目前已经开发出一些技术，可以通过工程aaRS催化氨基酸类似物对tRNA的酰化，从而促进新的侧链在体内整合到重组蛋白中。[4,5]本文中，我们描述了大肠杆菌苯丙酰- trna合成酶（Ala2943Gly；称为PheRS*）的突变形式的详细使用，该突变形式具有扩大的底物结合袋[4]，并且已被证明可以将对溴苯丙氨酸(1)掺入细菌宿主中表达的重组蛋白中。[4c， 6]我们现在发现对碘-、对氰基-、对乙基-和对叠氮-苯丙氨酸（2±5）和2-、3-和4-吡啶基丙氨酸（7±9）也可以在配备PheRS*的细菌宿主中取代苯丙氨酸。大肠杆菌菌株AF-IQ [pQE-FS]是一种苯丙酸营养不良菌，含有一种编码PheRS*和小鼠二氢叶酸还原酶（DHFR）的质粒图1显示了在添加苯丙氨酸或类似物1±9的培养基中诱导DHFR表达后，10 mL AF-IQ [pQE-FS]培养物的细胞裂解物的SDS-PAGE分析。DHFR的表达在所有培养中都很明显，除了添加
Macromolecular chemistry faces a dichotomy. Chemists can prepare polymers with a wide variety of functional groups, but cannot attain the sequence-specificity and monodispersity of proteins and nucleic acids. Conversely, the chemical diversity of proteins is severely constrained by the small number of amino acids specified by the genetic code. Can we find ways to combine the diversity of synthetic polymer chemistry with the precision of protein biosynthesis? One approach is to enhance the capability of the protein biosynthetic apparatus to utilize monomers other than the twenty canonical amino acids.[1, 2] Particular attention has been focused on the aminoacyl-tRNA synthetases (aaRS), which conjugate amino acids to their cognate tRNAs. The specificity of tRNA charging is pivotal for ensuring the fidelity of translation of genetic information into protein sequence.[3] Techniques have been developed for engineering aaRS to catalyze acylation of tRNA by amino acid analogues, facilitating incorporation of novel side chains into recombinant proteins in vivo.[4, 5] Herein we describe the elaboration of the use of a mutant form of the Esherichia coli phenylalanyl-tRNA synthetase (Ala2943Gly; termed PheRS*), which has an enlarged substrate binding pocket [4] and which has been shown to effect incorporation of p-bromophenylalanine (1) into a recombinant protein expressed in a bacterial host.[4c, 6] We now find that p-iodo-, p-cyano-, pethynyl-, and p-azido-phenylalanine (2±5) and 2-, 3-, and 4-pyridylalanine (7±9) can also be substituted for Phe in bacterial hosts outfitted with PheRS*. The E. coli strain AF-IQ [pQE-FS] is a Phe auxotroph that harbors a plasmid encoding PheRS* and the test protein murine dihydrofolate reductase (DHFR).[6] Figure 1 shows sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of cell lysates from 10 mL cultures of AF-IQ [pQE-FS] following induction of DHFR expression in minimal media supplemented with Phe or with one of the analogues 1±9. Expression of DHFR is evident in all cultures except that supplemented with