Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Targeted drug delivery requires 'loading' drugs onto targeting proteins. Traditional technologies for loading drugs rely on chemical conjugation of drugs or drug carriers to targeting proteins. An alternative approach might rely on assembly of targeting complexes using a docking system that includes two components: a 'docking' tag fused to a targeting protein, and a 'payload' module containing an adapter protein for non-covalent binding to the docking tag. We describe here a fully humanized adapter/docking tag system based on non-covalent interaction between two fragments of human pancreatic RNase I. A 15 amino acid long N-terminal fragment of RNase I designed to serve as a docking tag, was fused to the N-terminus of human vascular endothelial growth factor that served as a targeting protein. An 18-125 and an 18-127 amino acid long fragments of RNase I were engineered, expressed and refolded into active conformations to serve as adapter proteins. Interactions between the targeting and adapter proteins were characterized using enzymatic analysis and surface plasmon resonance. Targeting DNA delivery complexes were assembled, characterized by dynamic light scattering, and found to be very effective in receptor-mediated DNA delivery.
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PMID:Humanized docking system for assembly of targeting drug delivery complexes. 1273 51

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.
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PMID:Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery. 1460 Feb 7

High-affinity interactions of two fragments of human RNase I (1-15-aa Hu-tag and 21-125-aa HuS adapter protein) can be used for assembly of targeting drug delivery complexes. In this approach, a targeting protein is expressed as a fusion protein with a 15-aa Hu-tag, while HuS is conjugated to a drug (or a drug carrier) creating a "payload" module, which is then bound noncovalently to the Hu-tag of the targeting protein. Although this approach eliminates chemical modifications of targeting proteins, the payload modules are still constructed by random cross-linking of drugs or drug carriers to an adapter protein that might lead to functional heterogeneity of the complexes. To avoid this problem, we engineered an adapter protein HuS(N88C) with an unpaired cysteine in position 88 that can be directly modified without interference with activity of assembled targeting complexes. HuS(N88C) binds Hu-tagged annexin V with K(D) of 50 +/- 6 nM, which is comparable to that of wild-type HuS. To demonstrate the utility of HuS(N88C) for developing uniform payload modules, we constructed a HuS(N88C)-lipid conjugate and inserted it into preformed liposomes loaded with a fluorescent dye. Targeting proteins, Hu-tagged vascular endothelial growth factor or Hu-tagged annexin V, were docked to liposomes decorated with HuS, and the assembled complexes delivered liposomes selectively to target cells.
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PMID:Adapter protein for site-specific conjugation of payloads for targeted drug delivery. 1536 55

Random conjugation of therapeutic or diagnostic payloads to targeting proteins generates functionally heterogeneous products. Conjugation of payloads to an adapter that binds to a peptide tag engineered into a targeting protein provides an alternative strategy. To progress into clinical development, an adapter/docking tag system should include humanized components and be stable in circulation. We describe here an adapter/docking tag system based on mutated fragments of human RNase I that spontaneously bind to each other and form a conjugate with a disulfide bond between complimentary cysteine residues. This self-assembled "dock and lock" system utilizes the previously described fusion C-tag, a 1-15 aa fragment of human RNase I with the R4C amino acid substitution, and a newly engineered adapter protein (Ad-C), a 21-127-aa fragment of human RNase I with the V118C substitution. Two vastly different C-tagged recombinant proteins, human vascular endothelial growth factor (VEGF) and a 254-aa long N-terminal fragment of anthrax lethal factor (LFn), retain functional activities after spontaneous conjugation of Ad-C to N-terminal or C-terminal C-tag, respectively. Ad-C modified with pegylated phospolipid and inserted into the lipid membrane of drug-loaded liposomes (Doxil) retained the ability to conjugate C-tagged proteins, yielding targeted liposomes decorated with functionally active proteins. To further optimize the system, we engineered an adapter with an additional cysteine residue at position 88 for site-specific modification, conjugated it to C-tagged VEGF, and labeled with a near-infrared fluorescent dye Cy5.5, yielding a unique functionally active probe for in vivo molecular imaging. We expect that this self-assembled "dock and lock" system will provide new opportunities for using functionally active proteins for biomedical purposes.
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PMID:Self-assembled "dock and lock" system for linking payloads to targeting proteins. 1684 97

We developed a strategy for covalent coupling of targeting proteins to liposomes decorated with a standard adapter protein. This strategy is based on "dock and lock" the interactions between two mutated fragments of human RNase I, a 1-15-aa fragment with the R4C amino acid substitution, (Cys-tag), and a 21-127-aa fragment with the V118C substitution, (Ad-C). Upon binding to each other, Cys-tag and Ad-C spontaneously form a disulfide bond between the complimentary 4C and 118C residues. Therefore, any targeting protein expressed with Cys-tag can be easily coupled to liposomes decorated with Ad-C. Here, we describe the preparation of Ad-liposomes followed by coupling them to two Cys-tagged targeted proteins, human vascular endothelial growth factor expressed with N-terminal Cys-tag, and a 254-aa long N-terminal fragment of anthrax lethal factor carrying C-terminal Cys-tag. Both proteins retain functional activity after coupling to Ad-C-decorated drug-loaded liposomes. We expect that our "dock and lock" strategy will open new opportunities for development of targeted therapeutic liposomes for research and clinical use.
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PMID:A "dock and lock" approach to preparation of targeted liposomes. 2007 86

We developed a strategy for covalent coupling of targeting proteins to liposomes decorated with a standard adapter protein. This strategy is based on "dock and lock" interactions between two mutated fragments of human RNase I, a 1-15 aa fragment with the R4C amino acid substitution (Cys-tag), and a 21-127-aa fragment with the V118C substitution, (Ad-C). Upon binding to each other, Cys-tag and Ad-C spontaneously form a disulfide bond between the complementary 4C and 118C residues. Therefore, any targeting protein expressed with Cys-tag can be easily coupled to liposomes decorated with Ad-C. Here we describe the preparation of Ad-liposomes followed by coupling them to two Cys-tagged targeted proteins, human vascular endothelial growth factor expressed with N-terminal Cys-tag and a 254-aa long N-terminal fragment of anthrax lethal factor carrying C-terminal Cys-tag. Both proteins retain functional activity after coupling to Ad-C-decorated drug-loaded liposomes. We expect that our "dock and lock" strategy will open new opportunities for development of targeted therapeutic liposomes for research and clinical use.
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PMID:A "Dock and Lock" Approach to Preparation of Targeted Liposomes. 2783 32