| Title: |
Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery |
| Authors: |
Gaynutdinov, Timur I., Myshkin, Eugene, Backer, Joseph M., Backer, Marina V. |
| Source: |
Protein Engineering Design and Selection; October 01, 2003, Vol. 16 Issue: 10 p771-775, 5p |
| Abstract: |
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. |
| Database: |
Supplemental Index |
