Protein Engineering vol. 16 no. 10 pp. 771-775, 2003
© 2003 Oxford University Press
Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery
1SibTech, Inc., Newington, CT 06111, USA and 2Rammelkamp Center for Research, Case Western Reserve University School of Medicine, Cleveland, OH 44109, USA
3 To whom correspondence should be addressed. e-mail: mbacker{at}sibtech.com
Assembled modular complexes for targeted drug delivery can be based on strong non-covalent interactions between a cargo module containing an adapter protein and a docking tag fused to a targeting protein. We have recently constructed a completely humanized adapter/docking tag system based on interactions between 15 amino acid (Hu-tag) and 110 amino acid (HuS) fragments of human ribonuclease I (RNase I). Although recombinant HuS can be expressed and refolded into a functionally active form, the purification procedure is cumbersome and expensive, and more importantly, it yields a significant proportion of improperly folded proteins. Here we describe engineering, high-yield expression, and purification of a chimeric bovine/human RNase (BH-RNase) comprising 1–29 N-terminal amino acids of bovine ribonuclease A and 30–127 amino acids of human RNase I. Unlike RNase I, the chimeric BH-RNase can be cleaved by either subtilisin or proteinase K between A20 and S21, providing a functionally active HuS. The HuS obtained from chimeric BH-RNase differs from wild-type HuS by an N24T substitution; therefore, we have reverted this substitution by mutating N24 to T24 in BH-RNase. This BH-RNase mutant can also be cleaved by subtilisin or proteinase K yielding wild-type HuS. The affinity of HuS obtained from BH-RNase to Hu-tag is approximately 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.