Engineering S-protein fragments of bovine ribonuclease A for targeted drug delivery Marina V. Backer, * Timur I. Gaynutdinov, Renee Aloise, Kristen Przekop, and Joseph M. Backer SibTech Inc., 705 North Mountain Road, Newington, CT 06111, USA Received 14 May 2002 Abstract 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 con- structed a mutant 16–124aa fragment of RNase A in which 122 ala is replaced with a cysteine residue. The mutant and the corre- sponding 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. Ó 2002 Elsevier Science (USA). All rights reserved. Limited digestion of bovine pancreatic RNase A with subtilisin results in two enzymatically inactive frag- ments: 1–20aa (S-peptide) and 21–124aa (S-protein). These two fragments form a stable complex ðK d  10 9 MÞ, known as RNase S, with complete res- toration of ribonuclease activity [1]. It was found that only fifteen N-terminal amino acids of RNase A (1–15aa fragment) are necessary for reconstitution of fully active RNase S [2]. Currently, this fragment is used as a fusion ‘‘S-tag’’ for detection and affinity purification of re- combinant proteins [3]. We have reported recently that high affinity of S- protein/S-peptide interaction can be used for assembly of modular molecular vehicles for targeted drug delivery [4,5]. We proposed to create ‘‘payload’’ modules by conjugating drug carriers to S-protein and then to ‘‘dock’’ them non-covalently to S-tagged targeting pro- teins (Fig. 1). This approach avoids direct crosslinking of drug carriers to targeting proteins and therefore preserves their ability to interact with corresponding receptors or cell surface antigens. However, random chemical conjugation of drug carriers to S-protein still creates heterogeneous payload modules with variable affinities to S-tagged targeting proteins. We reasoned that construction of a mutant S-protein with an un- paired cysteine available for site-specific chemical mod- ifications might overcome this problem. To the best of our knowledge, expression of the wild type or mutant recombinant S-proteins has never been reported. In this study we describe construction, ex- pression, and purification of two functionally active 16– 124aa fragments of bovine pancreatic RNase A: the wild type and the A122C mutant with 122 cys available for site-specific modification. Materials and methods Construction of expression plasmids The pT7-7/RNase A plasmid encoding 1–124 amino acids of the mature bovine pancreatic RNase A was provided by Dr. G. DÕAlessio (Napoli Federico II University, Naples, Italy). Six different S-protein frag- ments were amplified by PCR from the pT7-7/RNase A plasmid with sense primers introducing ATG codon immediately upstream of the coding sequences (Table 1). An antisense primer 5 0 -CTACACTGAAGCATCAAA Protein Expression and Purification 26 (2002) 455–461 www.academicpress.com * Corresponding author. Fax: 860-953-1317. E-mail address: mbacker@sibtech.com (M.V. Backer). 1046-5928/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1046-5928(02)00546-6