Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic <i>E. coli</i> in Minimal Medium
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 (...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-07-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/14/11779 |
| Summary: | 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 <i>E. coli</i> BL-21 and auxotrophic <i>E. coli</i> B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b<sub>5</sub> (b<sub>5</sub>M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic <i>E. coli</i>, 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 b<sub>5</sub>M46, 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. |
|---|---|
| ISSN: | 1661-6596 1422-0067 |