High affinity interaction between S-protein and S-peptide fragments of bovine pancreatic RNase A has been recently used for construction of molecular vehicles for targeted drug delivery. The vehicle is assembled as a complex of drug carrier conjugated S-protein with S-peptide-tagged targeting protein. To avoid random chemical crosslinking of drug carriers to S-protein, we constructed a mutant 16-124aa fragment of RNase A in which (122)ala is replaced with a cysteine residue. The mutant and the corresponding wild type fragments expressed in Escherichia coli are refolded into functional conformations only in the presence of S-peptide. After the removal of S-peptide, both fragments retain the ability to bind S-peptide and S-peptide-tagged proteins. The (122)cys residue in the mutant fragment is available for site-specific conjugation. (C) 2002 Elsevier Science (USA). All rights reserved.