Efficient expression and purification of recombinant MUTYH protein in Escherichia coli

Abstract

MUTYH is a DNA glycosylase involved in the excisional base repair (BER) process responsible for the recognition and removal of adenine improperly bonded to 8-oxoguanine. Pathogenic variants or impaired MUTYH function are associated with MUTYH-associated polyposis and an increased risk of colorectal cancer. Recombinant MUTYH production is necessary for biochemical and structural studies. In this study, we developed and optimized a protocol for the expression and purification of recombinant human MUTYH protein fused with the 6×His N-terminal label. Wild-type constructs were cloned into the pET-28c expression vector and expressed in Escherichia coli Rosetta (DE3), ArcticExpress(DE3), and SHuffle® T7 Express strains. Low-temperature induction and optimized IPTG concentrations improved protein solubility. Recombinant MUTYH was purified using Ni2⁺-affinity chromatography followed by affinity chromatography with heparin. SDS-PAGE and Western blot analyses confirmed a high level of expression and effective isolation of the recombinant protein, with a significant portion isolated in a soluble form. The optimized workflow provides a reproducible method for obtaining purified MUTYH, suitable for subsequent biochemical and structural studies. In the future, we plan to evaluate its enzymatic activity.