Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery

Assembled modular complexes for targeted drug delivery can bebased on strong non-covalent interactions between a cargo modulecontaining an adapter protein and a docking tag fused to a targetingprotein. We have recently constructed a completely humanizedadapter/docking tag system based on interactions between 15amino acid (Hu-tag) and 110 amino acid (HuS) fragments of humanribonuclease I (RNase I). Although recombinant HuS can be expressedand refolded into a functionally active form, the purificationprocedure is cumbersome and expensive, and more importantly,it yields a significant proportion of improperly folded proteins.Here we describe engineering, high-yield expression, and purificationof a chimeric bovine/human RNase (BH-RNase) comprising 1–29N-terminal amino acids of bovine ribonuclease A and 30–127amino acids of human RNase I. Unlike RNase I, the chimeric BH-RNasecan be cleaved by either subtilisin or proteinase K betweenA20 and S21, providing a functionally active HuS. The HuS obtainedfrom chimeric BH-RNase differs from wild-type HuS by an N24Tsubstitution; therefore, we have reverted this substitutionby mutating N24 to T24 in BH-RNase. This BH-RNase mutant canalso be cleaved by subtilisin or proteinase K yielding wild-typeHuS. The affinity of HuS obtained from BH-RNase to Hu-tag isapproximately five times higher than that for recombinant HuS,reflecting a higher percentage of properly folded proteins. Received June 9, 2003; revised August 4, 2003; accepted August 28, 2003.