Incorporation of trifluoroisoleucine into proteins in vivo
Incorporation of trifluoroisoleucine into proteins in vivo
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DOI：
10.1021/ja0298287
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发表时间：
2003-06-11
影响因子：
15
通讯作者：
Tirrell, DA
中科院分区：
文献类型：
--
作者：
Wang, P;Tang, Y;Tirrell, DA
合成了两种含氟异亮氨酸衍生物：D，L-2-氨基-3-三氟甲基戊酸（3 TFI，2）和D，L-2-氨基-5，5，5-三氟甲基戊酸（5 TFI，3）。将5 TFI掺入模型靶蛋白，小鼠二氢叶酸还原酶（mDHFR）中，在异亮氨酸营养缺陷型大肠杆菌宿主菌株中，所述宿主菌株悬浮在补充有5 TFI的消耗异亮氨酸的基本培养基中。通过胰蛋白酶肽分析和蛋白质产物的基质辅助激光解吸电离质谱（MALDI-MS）确认5 TFI的掺入。氨基酸分析表明，93%以上的编码异亮氨酸残基被5 TFI取代。通过大肠杆菌异亮氨酰-tRNA合成酶（HeIRS）测定5 TFI的活化速率，得到比异亮氨酸低134倍的特异性常数（k（cat）/K-m）。成功地将5 TFI引入细胞因子鼠白细胞介素-2（mIL-2）的编码异亮氨酸位置。激发50%最大增殖反应的氟化蛋白的浓度为3.87 ng/mL，比野生型mIL-2的浓度高约30%（EC 50 = 2.70 ng/mL）。氟化和野生型细胞因子的最大反应是相等的，表明氟化蛋白质可以折叠成稳定的功能结构。3 TFI未产生体内掺入重组蛋白的证据，也未产生体外IleRS激活的证据。
Two fluorinated derivatives of isoleucine: D,L-2-amino-3-trifluoromethyl pentanoic acid (3TFI, 2) and D,L-2-amino-5,5,5-trifluoro-3-methyl pentanoic acid (5TFI, 3) were prepared. 5TFI was incorporated into a model target protein, murine dihydrofolate reductase (mDHFR), in an isoleucine auxotrophic Escherichia coli host strain suspended in 5TFI-supplemented minimal medium depleted of isoleucine. Incorporation of 5TFI was confirmed by tryptic peptide analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of the protein product. Amino acid analysis showed that more than 93% of the encoded isoleucine residues were replaced by 5TFI. Measurement of the rate of activation of 5TFI by the E coli isoleucyl-tRNA synthetase (HeIRS) yielded a specificity constant (k(cat)/K-m) 134-fold lower than that for isoleucine. 5TFI was successfully introduced into the cytokine murine interleukin-2 (mIL-2) at the encoded isoleucine positions. The concentration of fluorinated protein that elicits 50% of the maximal proliferative response is 3.87 ng/mL, about 30% higher than that of wild-type mIL-2 (EC50 = 2.70 ng/mL). The maximal responses are equivalent for the fluorinated and wild-type cytokines, indicating that fluorinated proteins can fold into stable and functional structures. 3TFI yielded no evidence for in vivo incorporation into recombinant proteins, and no evidence for activation by IleRS in vitro.