Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications

Abstract

The natural genetic code only allows for 20 standard amino acids in protein translation, but genetic code reprogramming enables the incorporation of non-standard amino acids (NSAAs). Proteins containing NSAAs provide enhanced or novel properties and open diverse applications. With increased attention to the recent advancements in synthetic biology, various improved and novel methods have been developed to incorporate single and multiple distinct NSAAs into proteins. However, various challenges remain in regard to NSAA incorporation, such as low yield and misincorporation. In this review, we summarize the recent efforts to improve NSAA incorporation by utilizing orthogonal translational system optimization, cell-free protein synthesis, genomically recoded organisms, artificial codon boxes, quadruplet codons, and orthogonal ribosomes, before closing with a discussion of the emerging applications of NSAA incorporation.

Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

“…Recent efforts in orthogonal translation systems improvement combined with the application of genetically recoded organisms allowed to improve the overall efficiency of methodology and to produce a recombinant protein with single or multiple site-specifically incorporated NAA with the yield equal or comparable with wild type protein synthesis. The intensive studies of the expanded genetic code methodology allowed not only for the development of highly effective tools for the protein molecular imaging, site-specific labelling, production of biomaterials or therapeutical peptide with improved and new properties [113], but also revealed that many other aspects must be considered as a potential limiting factor for the efficient NAA incorporation into recombinant proteins. For instance, the NAAs with bulky or extremely charged chemical group are unable to effectively cross cellular membrane, thus, the successful approach to evolve orthogonal pairs and site-specifically incorporate such a NAAs would be either propeptide strategy utilizing NAAs in dipeptide form [114] or modification of the host organism transport system, such as engineering of periplasmic binding protein, for optimized uptake of the desired NAA into the cell [115].…”

Exploring protein phosphorylation by combining computational approaches and biochemical methods

“…Protein expression with this method occurs in an open reaction environment to which crude cell extracts containing an overexpressed tRNA/aa-RS pair are added along with the ncAA, which is perpetually accessible to the translation machinery. There are several reports of increased production yield due to the implementation of CFPS protocols, as corroborated by western blot and mass spectrometry analyses [78] , [79] . Oza et al [80] developed a CFPS-based method to enhance the synthesis efficiency of site-specific Ser-phosphorylated constructs.…”

Engineered Cationic Antimicrobial Peptides (eCAPs) to Combat Multidrug-Resistant Bacteria

“…Since their initial discoveries in the 1980s [ 8 , 9 , 10 ], AMPs have been shown to occur in almost all life forms as short peptides (10–50 amino acids long) with an amphipathic (e.g., cationic or anionic and hydrophobic domains) structure in the context of host defense [ 11 , 12 , 13 , 14 ]. Although classical AMPs are ribosomally synthesized (composed of the standard proteinogenic amino acids) [ 15 , 16 ], AMPs can be extended to include well-known peptides that display a cationic (or the less commonly known anionic) amphipathic structure (e.g., the cationic lipopeptide polymyxins or the anionic daptomycin) [ 17 , 18 , 19 ]. Hence, ubiquitous in nature, AMPs represent the first line of defense against a variety of microbial pathogens (e.g., bacteria, fungi, parasites, viruses), depending on amino acid composition of the amphipathic motif [ 20 , 21 , 22 , 23 , 24 ].…”