title: Adapter protein for site-specific conjugation of payloads for targeted drug delivery.
abstract: 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.
authors.affiliation: Rammelkamp Center for Research, Case Western Reserve University School of Medicine, Cleveland, Ohio 44109, USA. mbacker@sibtech.com
authors.forename: Marina V
authors.initials: MV
authors.lastname: Backer
authors.forename: Timur I
authors.initials: TI
authors.lastname: Gaynutdinov
authors.forename: Vimal
authors.initials: V
authors.lastname: Patel
authors.forename: Brian T
authors.initials: BT
authors.lastname: Jehning
authors.forename: Eugene
authors.initials: E
authors.lastname: Myshkin
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Backer
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002461:Cell Line, Transformed; D004337:Drug Carriers / Q000737:chemistry* / Q000378:metabolism*; D016503:Drug Delivery Systems / Q000379:methods*; D006801:Humans; D008081:Liposomes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004337:Drug Carriers; D008081:Liposomes; D012259:Ribonuclease, Pancreatic