title: Preparation and functional evaluation of RGD-modified streptavidin targeting to integrin-expressing melanoma cells.
abstract: The vertical growth stage is the most dangerous stage of melanoma and is often associated with a poor prognosis. The increased invasiveness and metastasis that is typical for vertically growing melanoma are mediated by the molecules of cell adhesion (particularly, integrins). Integrin αvβ3, which is abundantly expressed on melanoma cells with high metastatic potentials and is characterized by low expression levels in normal melanocytes, is potentially an attractive target for melanoma diagnostics and therapy. Integrin αvβ3 is known to recognize the arginine-glycine-aspartic (RGD) sequence, which has been found in a wide variety of its natural ligands. Here expression vectors bearing the genes of fusion proteins have been constructed for producing these proteins in Escherichia coli. Such fusion proteins consist of a peptidic 'address,' targeting the integrins on melanoma cells, linked to an 'adaptor' for the attachment of a diagnostic or toxic agent. The peptidic 'address' contains the RGD motif, which is stabilized by a disulfide bond to achieve the optimal receptor binding conformation. The 'adaptor' is a tetrameric protein, namely, streptavidin, that is able to achieve high-affinity binding of d-biotin (K(d) = 10(-15) M) and confer avidity to the address peptide. This binding ability facilitates the generation of anti-melanoma diagnostic and therapeutic agents using the appropriate biotin derivatives. These recombinant proteins were purified from the periplasm of E.coli using columns with 2-iminobiotin agarose and demonstrated an ability to adhere to the surface of murine and human melanoma cells.
authors.affiliation: A.N. Bakh Institute of Biochemistry RAS, Leninsky Prospect, 33, Bld. 2, 119071, Moscow, Russia. krimsy@yandex.ru
authors.forename: Marina S
authors.initials: MS
authors.lastname: Syrkina
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Shirokov
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Rubtsov
authors.forename: Elena L
authors.initials: EL
authors.lastname: Kadyrova
authors.forename: Vladimir P
authors.initials: VP
authors.lastname: Veiko
authors.forename: Valentin A
authors.initials: VA
authors.lastname: Manuvera
mesh_terms: D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D004765:Enteropeptidase / Q000737:chemistry; D004926:Escherichia coli; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008297:Male; D008545:Melanoma; D051379:Mice; D009842:Oligopeptides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013194:Staining and Labeling; D019809:Streptavidin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C080396:trypsinogen activation peptide; C047981:arginyl-glycyl-aspartic acid; D019809:Streptavidin; D004765:Enteropeptidase; D016650:Fluorescein-5-isothiocyanate