Refined methodology to purify mucins from pig colonic mucosa

Mucin, the main macromolecular component of mucus, contains a peptide backbone rich in proline, threonine and serine (PTS) and oligosaccharide side chains. Glycosylation increases the molecular size of mucins and restricts access of proteases to mucin. Based on these characteristics, Faure et al. [Faure, M., Moënnoz, D., Montigon, F., Fay, L.B., Breuille, D., Finot, P.A., Ballèvre, O. and J. Boza. 2002. Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats. Anal. Biochem 307: 244–251.] developed a method to purify mucins from intestinal mucosa. In this method, mucins are reduced (RD) and digested with protease (dig) prior to isolation using size-exclusion chromatography. Our objective was to refine this method to purify mucins from pig colonic mucosa. Mucosal scrapings were homogenized in 5 mM EDTA and were either treated with 7 μL of protease inhibitor cocktail (PIC; UndigUnRD) or incubated with 100 μL of Protease at 37 °C for 80 min and then subsequently treated with PIC (digUnRD). Homogenates were fractionated based on size using a Sepharose CL-4B column. Mucin-containing fractions were identified by a combination of SDS-PAGE and staining techniques and were pooled. Mucin purified using digUnRD method had higher proportions of PTS compared to crude mucin purified using UndigUnRD method (31.9 vs. 23.5 mol/100 mol AA; P < 0.005). The proportion of PTS in mucin purified using digUnRD was also higher than mucin purified using digRD (31.9 vs. 25.7 mol/100 mol AA; P < 0.05). The refined method employing protease digestion and no reduction can be successfully applied to purify mucins from pig colonic mucosa.