Abstract
The diversity of non-canonical amino acids (ncAAs) endows proteins with new features for a variety of biological studies and biotechnological applications. The genetic code expansion strategy, which co-translationally incorporates ncAAs into specific sites of target proteins, has been applied in many organisms. However, there have been only few studies on pathogens using genetic code expansion. Here, we introduce this technique into the human pathogen Salmonella by incorporating p-azido-phenylalanine, benzoyl-phenylalanine, acetyl-lysine, and phosphoserine into selected Salmonella proteins including a microcompartment shell protein (PduA), a type III secretion effector protein (SteA), and a metabolic enzyme (malate dehydrogenase), and demonstrate practical applications of genetic code expansion in protein labeling, photocrosslinking, and post-translational modification studies in Salmonella. This work will provide powerful tools for a wide range of studies on Salmonella.
Highlights
Non-canonical amino acids are powerful tools for protein studies
In Escherichia coli, three pairs have been most successful: (1) an evolved orthogonal pair based on the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase and its cognate tRNA, which has been used to install a diverse array of tyrosine and phenylalanine derivatives[15]; (2) a natural orthogonal pair of pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcinaceae species, which facilitates the incorporation of a variety of lysine and phenylalanine analogs[16,17]; and (3) the phosphoseryl-tRNA synthetase (SepRS) and its cognate tRNA from methanogenic archaea, which were engineered for phosphoserine incorporation[18,19]
The expression of superfolder green fluorescent protein (sfGFP) in the pBAD vector can be modulated by the concentration of arabinose in the media (Supplementary Fig. S1)
Summary
Non-canonical amino acids (ncAAs) are powerful tools for protein studies. In the past few years, more than 150 different ncAAs have been incorporated into proteins in both prokaryotic and eukaryotic organisms using varied approaches. In Escherichia coli, three pairs have been most successful: (1) an evolved orthogonal pair based on the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (mjTyrRS) and its cognate tRNA, which has been used to install a diverse array of tyrosine and phenylalanine derivatives[15]; (2) a natural orthogonal pair of pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcinaceae species, which facilitates the incorporation of a variety of lysine and phenylalanine analogs[16,17]; and (3) the phosphoseryl-tRNA synthetase (SepRS) and its cognate tRNA from methanogenic archaea, which were engineered for phosphoserine incorporation[18,19] Salmonella infects both human and animals, causing millions of illnesses every year. We incorporated these ncAAs into Salmonella native proteins including a microcompartment shell protein, a type III secretion effector protein, and a TCA cycle enzyme respectively to demonstrate practical applications of genetic code expansion in protein labeling, photocrosslinking, and post-translational modification studies in Salmonella
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