Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery.
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.
SibTech, Inc., Newington, CT 06111, USA.
Timur I
TI
Gaynutdinov
Eugene
E
Myshkin
Joseph M
JM
Backer
Marina V
MV
Backer
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001430:Bacteriocins; D002417:Cattle; D002846:Chromatography, Affinity; D016503:Drug Delivery Systems; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D001426:Bacterial Proteins; D001430:Bacteriocins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D012259:Ribonuclease, Pancreatic; D019286:Endopeptidase K; C002836:subtilin