Enzymatic digestion as a tool for removing proteinaceous templates from molecularly imprinted polymers

Proteinaceous templates are often immobilized prior to polymerization in molecular imprinting which usually entails the need for digestion as a tool for subsequent template removal. The efficiency of digestion, however, has never been investigated in detail in such a context despite the well-known importance of the template removal step in creating selective binding sites. We have demonstrated that native proteins are often not efficiently cleaved by proteinase K, a highly efficient protease enzyme that can digest even keratin. We have studied and optimized the digestion conditions of a model protein, horseradish peroxidase (HRP), by comparing the obtained fragments to those predicted by in silico digestion. The highest cleaving efficiency was obtained after denaturation of the protein with a surfactant and reduction of its disulphide bridges. The protocol developed with HRP was also tested on avidin and was demonstrated to be applicable for template removal from HRP-or avidin-imprinted polymers.