Expanding the genetic code of Salmonella with non-canonical amino acids

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.

Genetically encoding thioacetyl‐lysine as a non‐deacetylatable analog of lysine acetylation in Escherichia coli

“…The procedure has small modifications from previous protocols . The genes of target proteins were cloned into the pBAD plasmid with a C‐terminal His 6 ‐tag, and transformed into Top10 cells together with the pTech plasmid harboring genes of tRNA Pyl and TAcKRS for expression.…”

“…The procedure has small modifications from the previous protocol . The purified proteins were trypsin digested by a standard in‐gel digestion protocol, and analyzed by LC‐MS/MS on an LTQ Orbitrap XL (Thermo Fisher Scientific) equipped with a nanoACQUITY UPLC system (Waters, Milford, MA, USA).…”

An inside look at a biofilm: Pseudomonas aeruginosa flagella bio-tracking

“…Therefore, it is necessary to To date, live-cell imaging can be obtained through different approaches, however genetic code expansion, the reassignment of codons and incorporation of an unnatural amino acid (Uaa) into proteins 17 , displays advantages over other methodologies, and is gaining increasing exposure and momentum [18][19][20][21] . Genetic code expansion systems are being constantly improved, expanded and adapted to a growing number of organisms [22][23][24][25] . This technique aids in improving imaging.…”

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

“…These features simplify the engineering of PylRS and TyrRS for a desired ncAA by in vivo selection methods (Figure 1c) and facilitate the targeting of different codons for reassignment (Table 1). The M. jannaschii TyrRS•tRNA Tyr pair is used in bacteria such as E. coli [1 •• ], Salmonella [9], Streptomyces [10], and Mycobacterium [11] (Table 1); yet this pair is not suitable for eukaryotic applications because identity elements overlap with eukaryotic TyrRS•tRNA Tyr . However, the E. coli TyrRS•tRNA Tyr pair has been adapted for GCE in diverse eukaryotic species [12]; although, the repertoire of ncAA substrates is smaller (Table 1).…”