Protein Chemical Modification Inside Living Cells Using Split Inteins

Abstract

Summary Methods to visualize, track, measure, and perturb or activate proteins in living cells are central to biomedical efforts to characterize and understand the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chemical labeling of proteins. We describe in this protocol the use of fluorescence resonance emission transfer (FRET)-quenched DnaE split-inteins for the site-specific labeling and concomitant fluorescence activation of proteins in living cells. We have successfully employed this approach for the site-specific in-cell labeling of the DNA binding domain (DBD) of the transcription factor YY1 using several human cell lines. Moreover, we have shown that this approach can be also used for modifying proteins in order to control their cellular localization and potentially alter their biological activity.

Protein Splicing of Inteins: A Powerful Tool in Synthetic Biology

“…Inteins were also applied in making recombinant C-terminal polypeptide α-thioesters, which were the crucial components for the semi-synthesis of chemically modified proteins using expressed protein ligation (EPL) ( Flavell and Muir, 2009 ; Fong and Wood, 2010 ; Li, 2015 ; Sarmiento and Camarero, 2019 ). Protein trans -splicing mediated by split inteins (naturally-occurring or artificially designed) has been used in proteins modification, such as introducing the site-specific modification including phosphorylation, biotinylation, ubiquitination, glycosylation, and segmental isotopic labeling both in vitro and in cells ( Qin et al, 2002 ; Rak et al, 2003 ; Shogren-Knaak et al, 2003 ; Chacko et al, 2004 ; Durek et al, 2004 ; Flavell and Muir, 2009 ; Borra and Camarero, 2017 ; Debelouchina and Muir, 2017 ). Inteins have also been utilized in generating cyclic proteins, in which the split intein fragments were fused to both sides of a target protein, and the N- and C-terminus of a target protein were joined through the association and splicing of the pairs ( Topilina and Mills, 2014 ; Nanda et al, 2020 ).…”

“…Chemical modification of proteins holds great potential for therapeutics engineering since it can help to understand the pharmacology and improve the property and effect of the drugs ( Wold, 1981 ; Borra and Camarero, 2017 ). Incorporating non-canonical amino acids (ncAAs) in a site-specific manner can partially address the issue ( Liu and Schultz, 2010 ).…”

Nanomanufacturing of biopolymers using electron and ion beams

“…Multi-level functionalization, employing genetic engineering [30][31][32], chemical modification [33,34], molecular doping [35] and solution mixing [36], can tune a wide range of properties in bionanostructures (figure 1, bottom left). (1) Thus, synthetic spider silk protein was designed and expressed in Escherichia coli through genetic engineering [37].…”