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.
Biochemické děje jako fotosyntéza a respirace, při nichž dochází k redoxním reakcím a přenosu elektronů pomocí kaskád proteinových molekul, souhrnně je označujeme jako elektron‐transportní systémy, jsou pro život na Zemi klíčové a nezbytné. Vhodným modelovým proteinem pro studium strukturně funkčních vztahů elektron‐transportních systémů proteinové povahy a vlivu aminokyselinových zbytků či záměny centrálního kovu na redox procesy je azurin, který patří do rodiny malých rozpustných cupredoxinů a podílí se na přenosu elektronů při anaerobní respiraci bakterií rodu Pseudomonas.
Biochemical processes like photosynthesis and cellular respiration, which involve redox reactions and are facilitated by electron transfer through a cascade of protein molecules, commonly referred to as electron‐transfer systems, play a key role in all life on Earth. Azurin is a suitable model to study structural properties of such electron‐transfer systems and the role of individual amino acid residues or the central metal ion in redox reactions. This protein belongs to the family of small soluble electron transporters known as cupredoxines and has a physiological role during anaerobic respiration in Pseudomonas bacterial species.
- Klíčová slova
- cupredoxiny,
- MeSH
- azurin * chemie MeSH
- transport elektronů MeSH
