Denaturing and reducing agents improve porcine gastric mucin purification while preserving functional properties

Research on mucus and its key macromolecular component, mucin glycoproteins, has gained considerable attention during the past decade. However, advances in this area are often limited by the fact that commercially available mucins are either not fully functional or come with a low degree of purity. Lab-extracted mucins, in contrast, have been shown to be highly functional, but controlling their level of purity remains critical since mucin-associated proteins are still present after the extraction process. We here modify an existing mucin extraction protocol for stomach mucus, which is based on filtration and size exclusion chromatography; our goal is to enhance the detachment of molecular contaminants from the mucins during the purification routine. In particular, the addition of a denaturing agent (guanidinium chloride, GuHCl) or a reducing agent (dithiothreitol, DTT) at specific steps of the purification cascade is shown to be efficient in removing contaminants attached to the mucin molecule through different molecular interactions: whereas the denaturing agent is more effective in removing physically bound proteins, the reducing agent maximizes the removal of small molecules covalently linked to the mucins. In addition, we find that the lubricating properties of mucins extracted in the presence of the chemical additives are maintained and – in the case of GuHCl addition – the anti-viral efficacy towards HIV is even enhanced, without negatively affecting the chemical architecture of mucin. Importantly, we show how choosing the right dose of the additive as well as the correct position within the sequence of purification steps is crucial to obtain this improved outcome.