posted on 2023-12-22, 20:33authored byAnastasiia
G. Tarabarova, Anton Lopukhov, Alexey N. Fedorov, Maria S. Yurkova
His-tags are protein affinity tags ubiquitously used
due to their
convenience and effectiveness. However, in some individual cases,
the attachment of His-tags to a protein’s N- or C-termini resulted
in impairment of the protein’s structure or function, which
led to attempts to include His-tags inside of polypeptide chains.
In this work, we describe newly designed internal His-tags, where
two triplets of histidine residues are separated by glycine residues
to avoid steric hindrances and consequently minimize their impact
on the protein structure. The applicability of these His-tags was
tested with eGFP, a multifaceted reference protein, and GrAD207, a
modified apical domain of GroEL chaperone, designed to stabilize in
soluble form initially insoluble proteins. Both proteins are used
as fusion partners for different purposes, and providing them with
His-tags introduced into their polypeptide chains should conveniently
broaden their functionality without involving the termini. We conclude
that the insertable tags may be adjusted for the purification of proteins
belonging to different structural classes.