Efficient Incorporation of Clickable Unnatural Amino Acids Enables Rapid and Biocompatible Labeling of Proteins in Vitro and in Bacteria

Abstract

Figure 1. Chemical structure of the uAA 4-propargyloxy-l-phenylalanine (pPR).

Tuning the Properties of Protein-Based Polymers Using High-Performance Orthogonal Translation Systems for the Incorporation of Aromatic Non-Canonical Amino Acids

“…We first profiled the substrate specificities of our previously selected aaRS variants: pAcFRS.1.t1, pAcFRS.2.t1, and pAzFRS.2.t1 were originally selected for pAcF and pAzF incorporation ( Amiram et al, 2015 ), MutRS-1—MutRS-4 were selected for pPR incorporation ( Hadar et al, 2021 ), and AzoRS-1-AzoRS-4 were selected for AzoPhe incorporation ( Israeli et al, 2021 ). A summary of the mutations in the AA binding site of these aaRS variants, as compared with the parent MjTyrRS, which natively recognizes tyrosine, is provided in Table 1 .…”

“…Notably, some high-performance aaRS variants, particularly pAcFRS.1.t1 and pAzFRS.2.t1, also exhibit a relatively high incorporation of natural AAs in the absence of their cognate ncAA. This property results in high levels of “background” expression of the ELP-GFP reporters in the absence of a ncAA, although, when present, they accurately incorporate their cognate ncAA ( Amiram et al, 2015 ; Hadar et al, 2021 ; Israeli et al, 2021 ). Such background expression can be reduced in conditions that highlight the differences in aaRS efficiencies, such as by increasing the number of TAG codons in the reporter protein, since the aaRSs incorporate the ncAAs more efficiently than the natural AAs.…”

“…We recently developed an aaRS evolution platform that utilizes multiplex automated genome engineering (MAGE) to create a library of chromosomal MjTyrRS variants. High-performance aaRS variants were selected from these libraries, enabling multi-site incorporation (10–30 instances per protein) of the aromatic ncAAs p-acetyl-phenylalanine (pAcF) ( Amiram et al, 2015 ), p-azido-phenylalanine (pAzF) ( Amiram et al, 2015 ), 4-propargyloxy- L -phenylalanine (pPR) ( Hadar et al, 2021 ), and phenylalanine-4‘-azobenzene (AzoPhe) ( Israeli et al, 2021 ). Although these aaRS variants were selected based on their ability to incorporate the ncAAs mentioned above, it had been previously demonstrated by us and others that most aaRS variants exhibit some degree of poly-specificity, that is, they can recognize and charge their cognate tRNA with ncAAs bearing various chemical groups ( Young et al, 2011 ; Amiram et al, 2015 ).…”

Conjugates of Recombinant Protein‐Based Polymers: Combining Precision with Chemical Diversity

“…Recent advances in protein evolution and the availability of specialized bacterial hosts now enable high‐yield, efficient incorporation of multiple instances of various uAAs including keto–, azide–, and alkyne‐bearing uAAs into recombinant proteins and PBPs produced in bacterial hosts. [ 22 ]…”

“…In addition, it was demonstrated that phase transition is retained upon fusion to other peptides or upon fabrication of ELP‐conjugate [27a,28] . A variety of the above‐mentioned conjugation strategies has been applied toward the conjugation of ELPs at precise locations on the protein termini or within the protein sequence by the addition of reactive AAs (i.e., cysteine, lysine, and tyrosine residues, which are absent from typical ELP sequences), [ 29 ] by the incorporation of uAAs, [22a,27c,30] or by the enzymatic modification of peptide motifs introduced into the ELP. [ 31 ] In the following, we elaborate on the advantages, utility, and novel properties enabled by ELP conjugation with various types of molecules ( Figure 2 ).…”