Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β)

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like alpha-subunit and a membrane-tethered growth factor-like beta-subunit. Due to the presence of several functional domains, the characterization of MUC4 beta is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4 beta (rMUC4 beta). Purified rMUC4 beta was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical beta-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4 beta physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4 beta that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.