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 E. coli BL-21 and auxotrophic E. coli B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b5 (b5M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic E. coli, 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 b5M46, 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.

• Keywords
• E. coli, azidohomoalanine, bio-orthogonal amino acid global substitution, homopropargylglycine, non-canonical amino-acid-containing proteins, photo-methionine,
• MeSH
• Alanine MeSH
• Amino Acids metabolism   MeSH
• Escherichia coli * genetics   metabolism   MeSH
• Methionine * metabolism   MeSH
• Proteins chemistry   MeSH
• Racemethionine metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Alanine MeSH
• Amino Acids MeSH
• azidohomoalanine MeSH Browser
• homopropargylglycine MeSH Browser
• Methionine * MeSH
• Proteins MeSH
• Racemethionine MeSH