Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Abstract

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, Na V 1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods.

Topography and motion of acid-sensing ion channel intracellular domains

“…While we consider this unlikely given the solubility of FPs, we cannot exclude this possibility. Future studies could employ smaller fluorophores such as the fluorescent non-canonical amino acid L-ANAP ( Chatterjee et al, 2013 ; Gordon et al, 2018 ; Gordon et al, 2016 ; Sakata et al, 2016 ; Zagotta et al, 2016 ) or split inteins and semi-synthesis ( Khoo et al, 2020 ; Lueck et al, 2016 ; Shah and Muir, 2014 ) to introduce small, bright fluorophores in specific sites. Such methods, ideally combined with acceptor groups ( Gordon et al, 2018 ; Gordon et al, 2016 ; Zagotta et al, 2016 ), will refine and test the provisional map we report here.…”

Topography and motion of the acid-sensing ion channel intracellular domains

“…The principle source of error is the possible disruption of the N or C tail caused by the addition or insertion of a fluorescent protein. Future studies could employ smaller fluorophores such as the fluorescent non-canonical amino acid L-ANAP 54-56 or split inteins and semi synthesis 57-59 to introduce small, bright fluorophores in specific sites. Such methods, ideally combined with acceptor groups 54-56 , will refine and test the provisional map we report here.…”

Advances and Opportunities in Epigenetic Chemical Biology

“…This method is particularly notable as the synthetic peptide can contain combinations of ncAAs, thus allowing a variety of labels and PTMs to be incorporated simultaneously. Further, because the modified peptide is generated synthetically and added to the cell, the heterogeneity and problems that can accompany GCE will not affect this system [401] . This technique will enable the study of protein PTMs in cellulo in a way that was previously inaccessible.…”