PMID:14600207
| Citation |
Gaynutdinov, TI, Myshkin, E, Backer, JM and Backer, MV (2003) Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery. Protein Eng. 16:771-5 |
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| 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. |
| Links | |
| Keywords |
Amino Acid Sequence; Animals; Bacterial Proteins; Bacteriocins; Cattle; Chromatography, Affinity; Drug Delivery Systems; Endopeptidase K/metabolism; Humans; Models, Molecular; Molecular Sequence Data; Mutation; Peptides/metabolism; Protein Binding; Protein Engineering; Protein Structure, Tertiary; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Ribonuclease, Pancreatic/chemistry; Ribonuclease, Pancreatic/genetics; Ribonuclease, Pancreatic/metabolism; Vascular Endothelial Growth Factor A/metabolism |
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