Abstract
Protein identification has gone beyond simply using protein/peptide tags and labeling canonical amino acids. Genetic code expansion has allowed residue- or site-specific incorporation of non-canonical amino acids into proteins. By taking advantage of the unique properties of non-canonical amino acids, we can identify spatiotemporal-specific protein states within living cells. Insertion of more than one non-canonical amino acid allows for selective labeling that can aid in the identification of weak or transient protein–protein interactions. This review will discuss recent studies applying genetic code expansion for protein labeling and identifying protein–protein interactions and offer considerations for future work in expanding genetic code expansion methods.
Highlights
Heterogeneous environments exist within living cells and this ever-changing environment affects the expression, dynamics, and state of proteins
Using genetic code expansion (GCE) to insert non-canonical amino acids in proteins of interest offers a high degree of specificity for downstream labeling in vitro and in vivo
SPI has allowed the incorporation of dozens of non-canonical amino acids (ncAAs) into cellular proteins and can be used for multiple investigative purposes, such as protein purification, determining cellular localization, and identifying interacting proteins (Lang and Chin, 2014)
Summary
Heterogeneous environments exist within living cells and this ever-changing environment affects the expression, dynamics, and state of proteins. Fixed cells from mammalian cell cultures and mouse models with newly synthesized proteins containing Aha or HPG are tagged with fluorescent dyes via click chemistry or can be combined with immunochemistry to visualize specific proteins The ease of inserting a tag encoding an ncAA led the authors to state the possibility of genomically inserting the modified tag to monitor endogenously expressed proteins This would ensure that any effects on protein folding and interactions would be minimized and the use of the tetrazine dyes allows live-cell imaging. Using genetically encoded crosslinkers offers a high degree of specificity that can capture strong and transient interactions
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