色氨酸
丝氨酸
吲哚试验
化学
色氨酸合酶
生物化学
突变体
异核单量子相干光谱
大肠杆菌
NS3型
氨基酸
分子生物学
立体化学
生物
核磁共振波谱
基因
蛋白酶
酶
作者
Elwy H. Abdelkader,Haocheng Qianzhu,Thomas Huber,Gottfried Otting
出处
期刊:ACS Sensors
[American Chemical Society]
日期:2023-10-27
卷期号:8 (11): 4402-4406
被引量:3
标识
DOI:10.1021/acssensors.3c01904
摘要
Genetic encoding of a noncanonical amino acid (ncAA) in an in vivo expression system requires an aminoacyl-tRNA synthetase that specifically recognizes the ncAA, while the ncAA must not be recognized by the canonical protein expression machinery. We succeeded in genetically encoding 7-aza-tryptophan (7AW), which is isoelectronic with tryptophan. The system is fully orthogonal to protein expression in Escherichia coli, enabling high-yielding site-selective isotope labeling in vivo. 7AW is readily synthesized from serine and 7-aza-indole using a tryptophan synthetase β-subunit (TrpB) mutant, affording easy access to isotope-labeled 7AW. Using labeled 7AW produced from 15N/13C-labeled serine, we produced 7AW mutants of the 25 kDa Zika virus NS2B-NS3 protease. 15N-HSQC spectra display single cross-peaks at chemical shifts near those observed for the wild-type protein labeled with 15N/13C-tryptophan, confirming the structural integrity of the protein and yielding straightforward NMR resonance assignments for site-specific probing.
科研通智能强力驱动
Strongly Powered by AbleSci AI