Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 43 Prague, Czech Republic.
Int J Mol Sci. 2023 Jul 22;24(14):11779. doi: 10.3390/ijms241411779.
Residue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (pMet), azidohomoalanine (Aha) and homopropargylglycine (Hpg), in prototrophic BL-21 and auxotrophic B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b (bM46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic , ncAA incorporation ranged from 50 to 70% for pMet and reached approximately 50% for Aha, after 26 h expression, with medium and low expression levels of MBP-GFP and bM46, respectively. In the prototrophic strain, by contrast, the protein expression levels were higher, albeit with a sharp decrease in the ncAA content after the first hours of expression. Similar expression levels and 70-80% incorporation rates were achieved in both bacterial strains with Hpg. Our findings provide guidance for expressing proteins with a high content of ncAAs, highlight pitfalls in determining the levels of methionine replacement by ncAAs by MALDI-TOF mass spectrometry and indicate a possible systematic bias in metabolic labeling techniques using Aha or Hpg.
非天然氨基酸（ncAAs）的残基特异性掺入可为蛋白质引入生物正交官能团。因此，该技术被应用于蛋白质的表征和定量。在这里，我们研究了在原养型 BL-21 和营养缺陷型 B834 中用三种蛋氨酸类似物（即光甲硫氨酸（pMet）、叠氮高丙氨酸（Aha）和同丙炔基甘氨酸（Hpg））表达蛋白质，以最大限度地提高 ncAA 含量，从而评估 ncAA 对细菌生长和细胞色素 b（bM46）、绿色荧光蛋白（MBP-GFP）和噬菌体休克蛋白 A 表达的影响。在营养缺陷型中，pMet 的 ncAA 掺入率在 26 小时表达后达到 50%至 70%，Aha 的 ncAA 掺入率约为 50%，而 MBP-GFP 和 bM46 的表达水平分别较低和中等。相比之下，在原养型菌株中，蛋白质表达水平更高，但在表达的前几个小时内 ncAA 含量急剧下降。在这两种细菌菌株中，Hpg 的表达水平和 70%-80%的掺入率相似。我们的研究结果为高 ncAA 含量的蛋白质表达提供了指导，强调了 MALDI-TOF 质谱法确定 ncAA 替代蛋氨酸水平的陷阱，并表明使用 Aha 或 Hpg 的代谢标记技术可能存在系统偏差。
