Open AccessHaloTag Forms an Intramolecular Disulfide
Author(s) -
Kirsten Deprey,
Joshua A. Kritzer
Publication year - 2021
Publication title -
bioconjugate chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.279
H-Index - 172
eISSN - 1520-4812
pISSN - 1043-1802
DOI - 10.1021/acs.bioconjchem.1c00113
Subject(s) - chemistry , cysteine , recombinant dna , extracellular , intramolecular force , intracellular , biophysics , disulfide bond , cytosol , biochemistry , stereochemistry , enzyme , biology , gene
HaloTag is a modified haloalkane dehalogenase used for many applications in chemical biology including protein purification, cell-based imaging, and cytosolic penetration assays. While working with purified, recombinant HaloTag protein, we discovered that HaloTag forms an internal disulfide bond under oxidizing conditions. In this work, we describe this internal disulfide formation and the conditions under which it occurs, and we identify the relevant cysteine residues. Further, we develop a mutant version of HaloTag, HaloTag8, that maintains activity while avoiding internal disulfide formation altogether. While there is no evidence that HaloTag is prone to disulfide formation in intracellular environments, researchers using recombinant HaloTag, HaloTag expressed on the cell surface, or HaloTag in the extracellular space might consider using HaloTag8 to avoid intramolecular disulfide formation.