| 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. |
| Publisher Information: |
Oxford University Press |
| Publication Year: |
2003 |
| Collection: |
HighWire Press (Stanford University) |
| Subject Terms: |
ORIGINAL ARTICLES |
| Description: |
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. |
| Document Type: |
text |
| File Description: |
text/html |
| Language: |
English |
| Relation: |
http://peds.oxfordjournals.org/cgi/content/short/16/10/771; http://dx.doi.org/10.1093/protein/gzg097 |
| DOI: |
10.1093/protein/gzg097 |
| Availability: |
http://peds.oxfordjournals.org/cgi/content/short/16/10/771; https://doi.org/10.1093/protein/gzg097 |
| Rights: |
Copyright (C) 2003, Oxford University Press |
| Accession Number: |
edsbas.5EDF7C90 |
| Database: |
BASE |