Incorporation of Non-Canonical Amino Acids into Proteins by Global Reassignment of Sense Codons

Abstract

Non-canonical amino acids are finding increasing use in basic and applied research. Proteins that evolved naturally for biological function did so by exploiting the chemistries of the canonical amino acids; however, when proteins are repurposed for biomedical and pharmacological applications, they are often subject to conditions different from those characteristic of their original biological environments. Non-canonical amino acids can impart properties that are inaccessible within canonical protein sequence space, and can thereby lead to improved or new functionality. We describe simple methods for global replacement of canonical amino acids by their non-canonical counterparts in recombinant proteins made in high yield in bacterial expression hosts. These methods can be used to engineer both chemical and physical properties of recombinant proteins.

Therapeutic promise of engineered nonsense suppressor tRNAs

“…GCE requires a “blank” codon that can be used as an incorporation signal for the ncAA. The most commonly used “blank” codon for GCE is traditionally the amber stop codon (UAG) because it is the least frequently used termination codon in the E. coli genome, although the other stop codons (Dumas et al, 2015), quadruplet codons (J. C. Anderson et al, 2004; Neumann et al, 2010), in some cases sense codons (K. Y. Fang et al, 2018; Schmitt et al, 2018), and even codon–anticodon pairs with noncanonical nucleotides (Feldman et al, 2019) have been employed for GCE.…”

Exploring protein phosphorylation by combining computational approaches and biochemical methods

“…The use of ncAAs in recombinant protein expression enables modification of the chemical properties of one or more residues [58] . Moreover, the use of technologies to selectively and co-translationally incorporate phosphomimetic amino acids into proteins can increase sample preparation yields to enable biophysical analysis.…”

“…Thus, recombinant expression of aa-RS/tRNA pairs and the protein construct of interest, together with the addition of the ncAA to the culture medium, constitute the basis for phosphomimetic protein biosynthesis [60] , [61] , [62] . The aa-RS/tRNA pair system has successfully been used with a wide variety of cells, both prokaryotic and eukaryotic, including yeast, plants and animals [58] . However, the effectiveness of ncAA incorporation into proteins is variable.…”

“…These few examples illustrate the convenience, even necessity, of optimizing the orthogonal translation systems. This can be achieved with high-yield protein expression vectors such as pEVOL, or selectivity-enhanced aa-RS, tRNA and/or bacterial elongation factor Tu mutants for stronger interactions [58] , [66] , [67] , [68] , [69] . It is also possible to use a knock-out of the tRNA competitor RF-1 for improved binding to amber/quadruplet codons, or engineered ribosomes for higher decoding efficiency [70] , [71] , [72] .…”

Two-Photon Absorption Cross-Sections in Fluorescent Proteins Containing Non-canonical Chromophores Using Polarizable QM/MM

“…The resulting chromophores from the incorporation of non-canonical amino acids are here called non-canonical chromophores (nCCs). Incorporation of nCCs has been accomplished and studied previously by experimental means as discussed for example by Fang et al (2018) and Budisa and Pal (2004). Regarding FPs, gold fluorescent protein (Bae et al, 2003) is an example of inclusion of non-canonical amino acids for which one-photon absorption properties have been measured experimentally and explored computationally, as will be discussed later in this text.…”