Production and Characterization of Fusion Proteins Containing Transferrin and Nerve Growth Factor

Park, E.; Starzyk, Ruth M.; McGrath, John P.; Lee, T.; George, Jonathan S.; Schütz, A; Lynch, P.D; Putney, Scott D.

doi:10.3109/10611869808997881

Cited by 15 publications

(8 citation statements)

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“…Others have also found that the insertion of IgG hinge region as a flexible linker between Tf and nerve growth factor (NGF) domains was ineffective for the expression of this fusion protein (12). To further demonstrate the requirement of the helical structure of the linker peptide for the increase of the fusion protein expression, we reversed the DNA sequence coding for the helical peptide to produce a linker with an identical peptide length, but a non-helical structure.…”

Section: Discussionmentioning

confidence: 99%

“…The selection of a peptide linker with the ability to maintain domain function of the fusion protein is becoming important (9–12). Recently, we designed a helical linker with 50 amino acids using an EAAAK helix-forming motif based on a previous study (10), and inserted the linker between granulocyte colony stimulating factor (G-CSF) and Tf moieties, leading to increased biological activity (13).…”

Section: Introductionmentioning

confidence: 99%

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Insertion of the Designed Helical Linker Led to Increased Expression of Tf-Based Fusion Proteins

2008

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Purpose To demonstrate a high-level expression of transferrin (Tf)-based fusion proteins by inserting a helical linker between two protein domains. Methods Tf-based fusion proteins were designed to contain oligonucleotides encoding a helical linker inserted between the protein domains. Plasmid constructs were transfected into HEK293 cells and the secreted fusion proteins were purified from conditioned serum free media. Expression was assessed using both SDS-PAGE and Western Blot using anti-hGH, G-CSF, or Tf antibodies; protein bands were analyzed using Quantity One software. The function of fusion proteins consisting of human growth hormone (hGH) and Tf was evaluated in Nb2 cell proliferation assays. Results The fusion proteins containing a helical linker, hGH-(H4)2-Tf and Tf-(H4)2-hGH, were expressed 1.7- and 2.4-fold higher, respectively, with a 2-fold lower ED50 than the hGH-Tf fusion protein without a helical linker. The Tf-(H4)2-G-CSF fusion protein exhibited a greater expression with an 11.2-fold increase compared with Tf-G-CSF fusion protein. Conclusions The helical linker introduced in Tf-fusion proteins resulted in a high-level of expression with improved in vitro bioactivity. This approach provides a simple method to increase poor expression of other fusion proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Insertion of the Designed Helical Linker Led to Increased Expression of Tf-Based Fusion Proteins

2008

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“…220 By designing conjugates between OX26 and therapeutic agents, such as low molecular drugs (methotrexate), neuropeptides (vasoactive intestinal peptide), polyamide nucleic acids, proteins (never growth factor NGF), which can be transcytosed via the transferrin receptor to the brain side of the blood-brain barrier, the effective concentration of drugs delivered to the brain has been markedly increased compared to the intravenous administration of the drugs alone. [221][222][223][224][225][226] In addition to the chemical conjugation, attempts have also been made to couple the therapeutics to the OX26 antibody using the avidin/biotin systems to promote coupling of biotin and biotinylated drugs to brain transport vectors. [227][228][229] A novel Ab-avidin fusion protein (Ab genetically fused to avidin) was constructed to deliver biotinylated compounds across the blood-brain barrier, and the fusion protein exhibited superior [ 3 H]biotin uptake into brain parenchyma in comparison with the chemical conjugate OX26-SA (Ab chemically conjugated to streptividin).…”

Section: Transferrin Receptors In Drug and Gene Delivery To The Brainmentioning

confidence: 99%

Transferrin/transferrin receptor‐mediated drug delivery

Qian

2002

Medicinal Research Reviews

409

247

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Since transferrin was discovered more than half a century ago, a considerable effort has been made towards understanding tranferrin-mediated iron uptake. However, it was not until recently with the identification and characterization of several new genes related to iron homeostasis, such as the hemochromatosis protein HFE and the iron transporter DMT1, that our knowledge has been advanced dramatically. A major pathway for cellular iron uptake is through internalization of the complex of iron-bound transferrin and the transferrin receptor, which is negatively modulated by HFE, a protein related to hereditary hemochromatosis. Iron is released from transferrin as the result of the acidic pH in endosome and then is transported to the cytosol by DMT1. The iron is then utilized as a cofactor by heme and ribonucleotide reductase or stored in ferritin. Apart from iron, many other metal ions of therapeutic and diagnostic interests can also bind to transferrin at the iron sites and their transferrin complexes can be recognized by many cells. Therefore, transferrin has been thought as a "delivery system" for many beneficial and harmful metal ions into the cells. Transferrin has also be widely applied as a targeting ligand in the active targeting of anticancer agents, proteins, and genes to primary proliferating malignant cells that overexpress transferrin receptors. This is achieved by conjugation of transferrin with drugs, proteins, hybride systems with marcomolecules and as liposomal-coated systems. Conjugates of anticancer drugs with transferrin can significantly improve the selectivity and toxicity and overcome drug resistance, thereby leading to a better treatment. The coupling of DNA to transferrin via a polycation such as polylysine or via cationic liposomes can target and transfer of the extrogenous DNA particularly into proliferating cells through receptor-mediated endocytosis. These kinds of non-viral vectors are potential alternatives to viral vectors for gene therapy, if the transfection efficiency can be improved. Moreover, transferrin receptors have shown potentials in delivery of therapeutic drugs or genes into the brain across blood-brain barrier.

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“…Fusion proteins have found applications in a wide spectrum of areas such as the biomedical field [including immunology, cancer research, and drug delivery (17)(18)(19)(20)] and materials science [self-assembled materials (e.g., gels), quantum dot bioconjugates, sensors, and inorganic materials synthesis (21)(22)(23)(24)(25)(26)(27)]. Here we describe new silica-based nanocomposites formed from bioengineered fusion proteins that consist of two components (Fig.…”

mentioning

confidence: 99%

Novel nanocomposites from spider silk–silica fusion (chimeric) proteins

Foo

Patwardhan

Belton

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

188

147

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Silica skeletal architectures in diatoms are characterized by remarkable morphological and nanostructural details. Silk proteins from spiders and silkworms form strong and intricate self-assembling fibrous biomaterials in nature. We combined the features of silk with biosilica through the design, synthesis, and characterization of a novel family of chimeric proteins for subsequent use in model materials forming reactions. The domains from the major ampullate spidroin 1 (MaSp1) protein of Nephila clavipes spider dragline silk provide control over structural and morphological details because it can be self-assembled through diverse processing methods including film casting and fiber electrospinning. Biosilica nanostructures in diatoms are formed in aqueous ambient conditions at neutral pH and low temperatures. The R5 peptide derived from the silaffin protein of Cylindrotheca fusiformis induces and regulates silica precipitation in the chimeric protein designs under similar ambient conditions. Whereas mineralization reactions performed in the presence of R5 peptide alone form silica particles with a size distribution of 0.5-10 m in diameter, reactions performed in the presence of the new fusion proteins generate nanocomposite materials containing silica particles with a narrower size distribution of 0.5-2 m in diameter. Furthermore, we demonstrate that composite morphology and structure could be regulated by controlling processing conditions to produce films and fibers. These results suggest that the chimeric protein provides new options for processing and control over silica particle sizes, important benefits for biomedical and specialty materials, particularly in light of the all aqueous processing and the nanocomposite features of these new materials.biomaterials ͉ nanostructures ͉ silaffin ͉ biomineralization ͉ ceramics C omplex mineralized composite systems in nature provide rich ground for insight into mechanisms of biomineralization and novel materials designs (1-4). Some of the more common sources of inspiration include seashells, insect exoskeletons, extracellular matrices involved in bone and other hard tissues, and biosilica skeletons. The formation of natural inorganic-organic composites is a multistep process, including the assembly of the extracellular matrix, the selective transportation of inorganic ions to discrete organized compartments with subsequent mineral nucleation, and growth delineated by preorganized cellular compartments. Silica skeletons found in nature are based on nanoscale composites wherein the organic components, usually proteins, are functional parts of the skeletal structures while also serving as silica-forming components (5, 6). As a result, materials' toughness is improved, strength is retained, and fine morphological control is achieved, all hallmark attributes of biological composites.Silica is widespread in biological systems and serves different functions, including support and protection in single-celled organisms, such as diatoms through to higher plants and animals (7,8...

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Production and Characterization of Fusion Proteins Containing Transferrin and Nerve Growth Factor

Cited by 15 publications

References 58 publications

Insertion of the Designed Helical Linker Led to Increased Expression of Tf-Based Fusion Proteins

Insertion of the Designed Helical Linker Led to Increased Expression of Tf-Based Fusion Proteins

Transferrin/transferrin receptor‐mediated drug delivery

Novel nanocomposites from spider silk–silica fusion (chimeric) proteins

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