Cloning and Characterization of Scavidin, a Fusion Protein for the Targeted Delivery of Biotinylated Molecules

Lehtolainen, Pauliina; Taskinen, Anna; Laukkanen, Johanna; Airenne, Kari J.; Heino, Sarika; Lappalainen, Maarit; Ojala, Kirsi; Marjomäki, Varpu; Martin, John F.; Kulomaa, Markku S.; Ylä‐Herttuala, Seppo

doi:10.1074/jbc.m109431200

Cited by 24 publications

(25 citation statements)

References 20 publications

(20 reference statements)

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“…28 In scavidin, it was anticipated that fusion protein forms trimers due to native characteristics of the trimeric scavenger receptor. 29 Instead, in crosslinking studies, the strongest signal was from a tetrameric form of the fusion protein.…”

Section: Targeting Of Biotinylated Compounds P Lehtolainen Et Almentioning

confidence: 99%

“…A sample of proteins was acetylated in situ prior to SDS-PAGE, 31 in order to detect higher order oligomers under nondenaturing conditions. 28 Supernatants were subjected to SDS-PAGE without boiling (nondenaturing conditions). A boiled sample (5 min) was used as an acetylation control (i.e.…”

Section: Western Blot and Crosslinking Studiesmentioning

confidence: 99%

“…28,32 Briefly, HRP (2 mg/ml) was added to the culture medium for 5 min after 4 h of fusion protein production in SFV-transduced cells. Postnuclear supernatant was prepared and mixed with a homogenate buffer containing Percoll (27%) in 0.25 M sucrose and BSA.…”

Section: Percoll Fractionationmentioning

confidence: 99%

“…The expression of the fusion protein was analyzed with antiavidin antibody staining as described earlier. 28,37 Samples were mounted in Mowiol (Calbiochem) and examined using a confocal microscope (Zeiss Microsystems; LSM-510 software with Photoshop 4.0).…”

Section: Fluorescence Microscopymentioning

confidence: 99%

“…BT4C rat glioma cells (10 000 cells/5 ml) were implanted intracranially in the right corpus callosum at a depth of 2.5 mm at the following coordinates: 1 mm caudal to bregma and 2 mm right to sutura sagittalis. 28,38 After 2 weeks of cell inoculations, Av-fusion protein/SFV virus in 0.9% NaCl, or empty SFV (control), was injected by Hamilton syringe with a 27-gauge needle into the tumor (see coordinates above) in 10 ml volumes, first into a depth of 2.5 mm during 2 min and then into a depth of 1.5 mm. To prevent the escape of the virus from the needle track, the syringe was held in place for 8 min.…”

Section: In Vivo Rat Malignant Glioma Model and Immunohistochemistrymentioning

confidence: 99%

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Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor–avidin fusion protein

et al. 2003

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The very high binding affinity of avidin to biotin is one of the highest to occur in nature. We constructed a fusion protein composed of avidin and the endocytotic LDL receptor in order to target biotinylated molecules to cells of the desired tissues. In addition to the native avidin, charge-mutated and nonglycosylated avidins were utilized as part of the fusion proteins, in order to modify its properties. All of the fusion protein versions retained the biotin-binding capacity. Although the specificity was not increased, however, fusion proteins composed of natural avidin and nonglycosylated avidin bound most efficiently to the biotinylated ligands. Fluorescence microscopy and atomic force microscopy studies revealed the expression of the fusion protein on cell membranes, and demonstrated specific and high-affinity binding of biotin to the low-density lipoprotein receptor (LDLR)-avidin fusion protein in vitro. Additionally, systemically administered biotinylated ligand targeted with high specificity the intracerebral tumors of rats that were expressing fusion protein after the virus-mediated gene transfer. These results suggest that local gene transfer of the fusion protein to target tissues may offer a novel tool for the delivery of biotinylated molecules in vitro and in vivo for therapeutic and imaging purposes.

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Section: Targeting Of Biotinylated Compounds P Lehtolainen Et Almentioning

confidence: 99%

Section: Western Blot and Crosslinking Studiesmentioning

confidence: 99%

Section: Percoll Fractionationmentioning

confidence: 99%

Section: Fluorescence Microscopymentioning

confidence: 99%

Section: In Vivo Rat Malignant Glioma Model and Immunohistochemistrymentioning

confidence: 99%

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Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor–avidin fusion protein

et al. 2003

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Efficient gene therapy based targeting system for the treatment of inoperable tumors

Wirth

Pikkarainen

Samaranayake

et al. 2012

The Journal of Gene Medicine

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Background A considerable percentage of tumors are not amenable to surgery. We have designed a simple and powerful targeting system that offers an alternative option for the multi‐component pre‐targeting strategies used clinically. This targeting system can be used for any type of solid tumors independent of the tumor type, thereby omitting the need to engineer unique antibodies for each specific application or tumour type. In the present study, we show the expression of a chimeric fusion protein, which contains the low‐density lipoprotein receptor transmembrane domains and avidin, after local gene transfer and its ability to bind biotinylated compounds in vivo. Methods Semliki Forest virus and lentivirus vectors were used to express the fusion protein with a high affinity binding site for biotinylated compounds in the tumor. Three different animal models and imaging modalities were used for the demonstration of the functionality and efficacy of the targeting system in vitro and in vivo. Results We demonstrate targeting of biotinylated compounds after local gene transfer in vivo using two different gene transfer vectors. The findings were confirmed by immunohistochemistry, single‐photon emission computed tomography and magnetic resonance imaging. The therapeutic efficacy was tested in a syngeneic rat glioma model by injecting biotinylated‐90Yttrium into the tail vein of glioma bearing rats. The study demonstrates that animals, which were treated by using the gene therapy based targeting system, lived significantly longer than control animals. Conclusions Our gene therapy based targeting system is a promising tool for the treatment of inoperable tumors and other disease conditions, as well as diagnostic imaging. Copyright © 2012 John Wiley & Sons, Ltd.

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Targeted magnetic resonance imaging of Scavidin‐receptor in human umbilical vein endothelial cells in vitro

Mantyla

Hakumäki

Huhtala

et al. 2006

Magnetic Resonance in Med

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Current therapeutic approaches to treat cancer are often hampered by the lack of specificity of the drugs used for therapy. Scavidin, a novel fusion protein expressed on cell membranes, could be utilized for targeting of therapeutic molecules. Scavidin exploits the high binding affinity between avidin and biotin and is capable of mediating endocytosis of a bound ligand. In the current study we evaluated the efficiency of biotinylated ultrasmall superparamagnetic iron oxide (

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Cloning and Characterization of Scavidin, a Fusion Protein for the Targeted Delivery of Biotinylated Molecules

Cited by 24 publications

References 20 publications

Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor–avidin fusion protein

Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor–avidin fusion protein

Efficient gene therapy based targeting system for the treatment of inoperable tumors

Targeted magnetic resonance imaging of Scavidin‐receptor in human umbilical vein endothelial cells in vitro

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