Recent studies indicate that enzymatic treatment of the infectious PrPSc prion under defined conditions could be an effective method to inactivate infectious prions. However, field studies on prion inactivation are hampered by restricted access to the dangerous and expensive infectious prion material. Hence, a surrogate marker for infectious prions would facilitate more practical prion inactivation research. Protein Sup35p, a non-pathogenic prion-like protein produced in yeast, has physical and chemical properties very similar to the BSE prion. Sup35NMHis6, a derivative of Sup35p, was produced from Escherichia coli by gene cloning, protein expression and purification. Monomeric Sup35NM-His6 is soluble. When aggregated, it forms prion-like amyloid, insoluble and resistant to proteases. Similar to BSE prion, a pre-heating step renders this protein digestible by proteinase K, subtilisin and keratinase but not collagenase and elastase. These results indicated that Sup35NM-His6, being simple and inexpensive to produce and non-pathogenic, can be a potential ideal candidate of prion surrogate protein in the study of prion inactivation and prevention of prion diseases. (c) 2004 Elsevier Inc. All rights reserved.