Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Colloid Interface Sci ; 581(Pt A): 218-225, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32771733

RESUMO

We used the Surface Forces Apparatus to elucidate the interaction mechanism between grafted 5 heptad-long peptides engineered to spontaneously form a heterodimeric coiled-coil complex. The results demonstrated that when intimate contact between peptides is reached, binding occurs first via weakly interacting but more mobile distal heptads, suggesting an induced-fit association process. Precise control of the distance between peptide-coated surfaces allowed to quantitatively monitor the evolution of their biding energy. The binding energy of the coiled-coil complex increased in a stepwise fashion rather than monotonically with the overlapping distance, each step corresponding to the interaction between a quantized number of heptads. Surface forces data were corroborated to surface plasmon resonance measurements and molecular dynamics simulations and allowed the calculation of the energetic contribution of each heptad within the coiled-coil complex.

2.
Biomacromolecules ; 20(5): 1926-1936, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-30951296

RESUMO

Affinity-based systems represent a promising solution to control the delivery of therapeutics using hydrogels. Here, we report a hybrid system that is based on the peptidic E/K coiled coil affinity pair to mediate the release of gold nanoparticles (NPs) from alginate scaffolds. On one hand, the gold NPs were functionalized with the Ecoil-tagged epidermal growth factor (EGF). The bioactivity of the grafted EGF and the bioavailability of the Ecoil moiety were confirmed by EGF receptor phosphorylation assays and by capturing the functionalized NPs on a Kcoil-derivatized surface. On the other hand, alginate chains were modified with azido-homoalanine Kcoil (Aha-Kcoil) by azide-alkyne click chemistry. The hybrid system was formed by dispersing NPs functionalized with the Ecoil-tagged EGF in alginate hydrogels containing either 0, 10, or 20% of Kcoil-modified alginate (Alg-Kcoil). With 20% of Alg-Kcoil, the release of Ecoil-functionalized NPs was reduced by half when compared to the release of NPs without Ecoil, whereas little to no differences were noticed with either 0 or 10% of Alg-Kcoil. Differential dynamic microscopy was used to determine the diffusion coefficient of the NPs, and the results showed a decrease in the diffusion coefficient of Ecoil-functionalized NPs, when compared to bare PEGylated NPs. Altogether, our data demonstrated that the E/K coiled coil system can control the release of NPs in a high Kcoil content alginate gel, opening diverse applications in drug delivery.

3.
Angew Chem Int Ed Engl ; 58(5): 1308-1314, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30426644

RESUMO

Demand for long-lasting antifouling surfaces has steered the development of accessible, novel, biocompatible and environmentally friendly materials. Inspired by lubricin (LUB), a component of mammalian synovial fluid with excellent antifouling properties, three block polymers offering stability, efficacy, and ease of use were designed. The bottlebrush-structured polymers adsorbed strongly on silica surfaces in less than 10 minutes by a simple drop casting or online exposure method and were extremely stable in high-salinity solutions and across a wide pH range. Antifouling properties against proteins and bacteria were evaluated with different techniques and ultralow fouling properties demonstrated. With serum albumin and lysozyme adsorption <0.2 ng cm-2 , the polymers were 50 and 25 times more effective than LUB and known ultralow fouling coatings. The antifouling properties were also tested under MPa compression pressures by direct force measurements using surface forces apparatus. The findings suggest that these polymers are among the most robust and efficient antifouling agents currently known.

4.
Bioconjug Chem ; 29(11): 3866-3876, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30350572

RESUMO

Thiol(-click) chemistry has been extensively investigated to conjugate (bio)molecules to polymers. Handling of cysteine-containing molecules may however be cumbersome, especially in the case of fast-oxidizing coiled-coil-forming peptides. In the present study, we investigated the practicality of a one-pot process to concomitantly reduce and conjugate an oxidized peptide to a polymer. Three thiol-based conjugation chemistries (vinyl sulfone (VS), maleimide, and pyridyldithiol) were assayed along with three reducing agents (tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol, and ß-mercaptoethanol). Seven out of the nine possible combinations significantly enhanced the conjugation yield, provided that an adequate concentration of reductant was used. Among them, the coincubation of an oxidized peptide with TCEP and a VS-modified polymer displayed the highest level of conjugation. Our results also provide insights into two topics that currently lack consensus: TCEP is stable in 10 mM phosphate buffered saline and it reacts with thiol-alkylating agents at submillimolar concentrations, and thus should be carefully used in order to avoid interference with thiol-based conjugation reactions.


Assuntos
Química Click/métodos , Peptídeos/química , Polímeros/química , Substâncias Redutoras/química , Compostos de Sulfidrila/química , Alquilação , Maleimidas/síntese química , Maleimidas/química , Oxirredução , Peptídeos/síntese química , Polímeros/síntese química , Substâncias Redutoras/síntese química , Compostos de Sulfidrila/síntese química , Sulfonas/síntese química , Sulfonas/química
5.
Biointerphases ; 12(1): 010501, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28325051

RESUMO

In an effort to rationalize and optimize an antiapoptotic coating combining chondroitin sulfate (CS) and epidermal growth factor (EGF) for vascular applications, the authors here report the comparison of two grafting strategies aiming to display EGF in an oriented fashion on CS. For that purpose, the authors produced, purified, and characterized a chimeric protein corresponding to EGF that was N-terminally fused to a cysteine and a coil peptide. The chimera was covalently immobilized via its free thiol group or captured via coiled-coil interactions at the surface of a biosensor or on a chondroitin sulfate coating in multiwell plates, mimicking the coating that was previously developed by them for stent-graft surfaces. The interactions of grafted EGF with the soluble domain of its receptor or the impact of grafted EGF upon vascular smooth muscle survival in proapoptotic conditions indicated that the coiled-coil based tethering was the best approach to display EGF. These results, combined to direct enzyme-linked immunosorbent assay measurements, indicated that the coiled-coil tethering approach allowed increasing the amount of bioavailable EGF when compared to covalent coupling, rather than the total amount of grafted EGF, while using much lower concentrations of tagged EGF during incubation.


Assuntos
Sulfatos de Condroitina/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacocinética , Proteínas Imobilizadas/metabolismo , Proteínas Imobilizadas/farmacocinética , Animais , Disponibilidade Biológica , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Ligação Proteica , Ratos
6.
Acta Biomater ; 50: 198-206, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28069507

RESUMO

In the field of tissue engineering, the tethering of growth factors to tissue scaffolds in an oriented manner can enhance their activity and increase their half-life. We chose to investigate the capture of the basic Fibroblast Growth Factor (bFGF) and the Epidermal Growth Factor (EGF) on a gelatin layer, as a model for the functionalization of collagen-based biomaterials. Our strategy relies on the use of two high affinity interactions, that is, the one between two distinct coil peptides as well as the one occurring between a collagen-binding domain (CBD) and gelatin. We expressed a chimeric protein to be used as an adaptor that comprises one of the coil peptides and a CBD derived from the human fibronectin. We proved that it has the ability to bind simultaneously to a gelatin substrate and to form a heterodimeric coiled-coil domain with recombinant growth factors being tagged with the complementary coil peptide. The tethering of the growth factors was characterized by ELISA and surface plasmon resonance-based biosensing. The bioactivity of the immobilized bFGF and EGF was evaluated by a human umbilical vein endothelial cell proliferation assay and a vascular smooth muscle cell survival assay. We found that the tethering of EGF preserved its mitogenic and anti-apoptotic activity. In the case of bFGF, when captured via our adaptor protein, changes in its natural mode of interaction with gelatin were observed. STATEMENT OF SIGNIFICANCE: In an effort to functionalize collagen/gelatin-based biomaterials with growth factors, we have designed an adaptor protein corresponding to a collagen-binding domain fused to a coil peptide. In our strategy, this adaptor protein captures growth factors being tagged with the partner coil peptide in a specific, stable and oriented manner. We have found that the tethering of the Epidermal Growth Factor preserved its mitogenic and anti-apoptotic activity. In the case of the basic Fibroblast Growth Factor, the captured growth factor remained bioactive although its tethering via this adaptor protein modified its natural mode of interaction with gelatin. Altogether this strategy is easily adaptable to the simultaneous tethering of various growth factors.


Assuntos
Materiais Biocompatíveis , Fator de Crescimento Epidérmico , Fator 2 de Crescimento de Fibroblastos , Fibronectinas , Gelatina , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Imobilizadas , Proteínas Recombinantes de Fusão , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibronectinas/química , Fibronectinas/farmacologia , Gelatina/química , Gelatina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Teste de Materiais/métodos , Domínios Proteicos , Proteínas Recombinantes de Fusão/farmacologia , Engenharia Tecidual/métodos
7.
Biomacromolecules ; 18(3): 965-975, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28122454

RESUMO

We have developed a heterodimeric coiled-coil system based on two complementary peptides, namely (EVSALEK)5 and (KVSALKE)5, or E and K, for the attachment of E-tagged biomolecules onto K-decorated biomaterials. We here explore two approaches to control the strength and the stability of the E/K coiled-coil complex, and thus its potential for the controlled release of biomolecules. Those are Leucine-to-Alanine mutations in the K peptide (4 peptides with 0 to 3 mutations) and multivalent presentation of the E peptide (6 bio-objects from monomeric to dimeric and n-meric). Using E-tagged growth factors and nanoparticles as models, SPR-based assays performed under continuous flow indicated that the release rate was strongly affected by both approaches independently, and that the strength of the capture could be finely tuned over a wide range (apparent dissociation constant from 0.12 pM to 270 nM). Further release assays carried out in well-plates showed that the multivalent presentation only had a significant influence in this setup since the wells were not rinsed under continuous flow.


Assuntos
Materiais Biocompatíveis/química , Preparações de Ação Retardada/química , Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Dimerização , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Moleculares , Mutação , Nanopartículas/química
8.
Tissue Eng Part B Rev ; 23(2): 163-182, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27824290

RESUMO

Collagen-based biomaterials are widely used in the field of tissue engineering; they can be loaded with biomolecules such as growth factors (GFs) to modulate the biological response of the host and thus improve its potential for regeneration. Recombinant chimeric GFs fused to a collagen-binding domain (CBD) have been reported to improve their bioavailability and the host response, especially when combined with an appropriate collagen-based biomaterial. This review first provides an extensive description of the various CBDs that have been fused to proteins, with a focus on the need for accurate characterization of their interaction with collagen. The second part of the review highlights the benefits of various CBD/GF fusion proteins that have been designed for wound healing and bone regeneration.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Colágeno/química , Colágeno/farmacologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Humanos , Domínios Proteicos
9.
Acta Biomater ; 37: 69-82, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27039978

RESUMO

UNLABELLED: Multifunctional constructs providing a proper environment for adhesion and growth of selected cell types are needed for most tissue engineering and regenerative medicine applications. In this context, vinylsulfone (VS)-modified dextran was proposed as a matrix featuring low-fouling properties as well as multiple versatile moieties. The displayed VS groups could indeed react with thiol, amine or hydroxyl groups, be it for surface grafting, crosslinking or subsequent tethering of biomolecules. In the present study, a library of dextran-VS was produced, grafted to aminated substrates and characterized in terms of degree of VS modification (%VS), cell-repelling properties and potential for the oriented grafting of cysteine-tagged peptides. As a bioactive coating of vascular implants, ECM peptides (e.g. RGD) as well as vascular endothelial growth factor (VEGF) were co-immobilized on one of the most suitable dextran-VS coating (%VS=ca. 50% of saccharides units). Both RGD and VEGF were efficiently tethered at high densities (ca. 1nmol/cm(2) and 50fmol/cm(2), respectively), and were able to promote endothelial cell adhesion as well as proliferation. The latter was enhanced to the same extent as with soluble VEGF and proved selective to endothelial cells over smooth muscle cells. Altogether, multiple biomolecules could be efficiently incorporated into a dextran-VS construct, while maintaining their respective biological activity. STATEMENT OF SIGNIFICANCE: This work addresses the need for multifunctional coatings and selective cell response inherent to many tissue engineering and regenerative medicine applications, for instance, vascular graft. More specifically, a library of dextrans was first generated through vinylsulfone (VS) modification. Thoroughly selected dextran-VS provided an ideal platform for unbiased study of cell response to covalently grafted biomolecules. Considering that processes such as healing and angiogenesis require multiple factors acting synergistically, vascular endothelial growth factor (VEGF) was then co-immobilized with the cell adhesive RGD peptide within our dextran coating through a relevant strategy featuring orientation and specificity. Altogether, both adhesive and proliferative cues could be incorporated into our construct with additive, if not synergetic, effects.


Assuntos
Adesivos/química , Materiais Revestidos Biocompatíveis/química , Dextranos/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Oligopeptídeos/química , Fator A de Crescimento do Endotélio Vascular/química , Proliferação de Células , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/classificação , Humanos
10.
Acta Biomater ; 29: 239-247, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26485166

RESUMO

UNLABELLED: Growth factors (GFs) are potent signaling molecules that act in a coordinated manner in physiological processes such as tissue healing or angiogenesis. Co-immobilizing GFs on materials while preserving their bioactivity still represents a major challenge in the field of tissue regeneration and bioactive implants. In this study, we explore the potential of an oriented immobilization technique based on two high affinity peptides, namely the Ecoil and Kcoil, to allow for the simultaneous capture of the epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) on a chondroitin sulfate coating. This glycosaminoglycan layer was selected as it promotes cell adhesion but reduces non-specific adsorption of plasma proteins. We demonstrate here that both Ecoil-tagged GFs can be successfully immobilized on chondroitin sulfate surfaces that had been pre-decorated with the Kcoil peptide. As shown by direct ELISA, changing the incubation concentration of the various GFs enabled to control their grafted amount. Moreover, cell survival studies with endothelial and smooth muscle cells confirmed that our oriented tethering strategy preserved GF bioactivity. Of salient interest, co-immobilizing EGF and VEGF led to better cell survival compared to each GF captured alone, suggesting a synergistic effect of these GFs. Altogether, these results demonstrate the potential of coiled-coil oriented GF tethering for the co-immobilization of macromolecules; it thus open the way to the generation of biomaterials surfaces with fine-tuned biological properties. STATEMENT OF SIGNIFICANCE: Growth factors are potent signaling molecules that act in a coordinated manner in physiological processes such as tissue healing or angiogenesis. Controlled coimmobilization of growth factors on biomaterials while preserving their bioactivity represents a major challenge in the field of tissue regeneration and bioactive implants. This study demonstrates the potential of an oriented immobilization technique based on two high affinity peptides to allow for the simultaneous capture of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). Our system allowed an efficient control on growth factor immobilization by adjusting the incubation concentrations of EGF and VEGF. Of salient interest, co-immobilizing of specific ratios of EGF and VEGF demonstrated a synergistic effect on cell survival compared to each GF captured alone.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Imobilizadas/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Sinergismo Farmacológico , Fator de Crescimento Epidérmico/agonistas , Fator de Crescimento Epidérmico/química , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas Imobilizadas/agonistas , Proteínas Imobilizadas/química , Fator A de Crescimento do Endotélio Vascular/agonistas , Fator A de Crescimento do Endotélio Vascular/química
11.
Biomacromolecules ; 16(11): 3445-54, 2015 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-26393367

RESUMO

Numerous strategies have been proposed to decorate biomaterials with growth factors (GFs) for tissue engineering applications; their practicability as clinical tools, however, remains uncertain. We previously presented two complementary amphipathic peptides, namely, E5 and K5, which could be utilized as tags to direct GF capture onto organic materials via E5/K5 coiled-coil interactions. We here investigated their potential as mediators of GF physical adsorption. Enzyme-linked immunosorbent assays highlighted that both electrostatic and hydrophobic interactions could contribute to the adsorption process, without interfering with the peptides propensity for coiled-coil interactions. E5-tagged vascular endothelial growth factor, in particular, was efficiently adsorbed to poly(allylamine)-functionalized polystyrene, was maintained in a bioactive state and was steadily liberated over several days with little initial burst. This simple immobilization procedure was successfully applied to poly(ethylene terephthalate) films. Altogether, our data demonstrated that coil-tag-directed adsorption is a tunable, versatile and straightforward strategy to decorate biomaterials with GFs.


Assuntos
Proteínas Imobilizadas/química , Peptídeos/química , Engenharia Tecidual/métodos , Fator A de Crescimento do Endotélio Vascular/química , Adsorção , Materiais Biocompatíveis/química , Proliferação de Células/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Interações Hidrofóbicas e Hidrofílicas , Poliaminas/química , Polietilenotereftalatos/química , Eletricidade Estática , Propriedades de Superfície
12.
Acta Biomater ; 9(6): 6806-13, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23485856

RESUMO

Chimeric growth factors may represent a powerful alternative to their natural counterparts for the functionalization of tissue-engineered scaffolds and applications in regenerative medicine. Their rational design should provide a simple, readily scalable production strategy while improving retention at the site of action. In that endeavor, we here report the synthesis of a chimeric protein corresponding to human vascular endothelial growth factor 165 being N-terminally fused to an E5 peptide tag (E5-VEGF). E5-VEGF was successfully expressed as a homodimer in mammalian cells. Following affinity purification, in vitro surface plasmon resonance biosensing and cell survival assays confirmed diffusible E5-VEGF ability to bind to its receptor ectodomains, while observed morphological phenotypes confirmed its anti-apoptotic features. Additional surface plasmon resonance assays highlighted that E5-VEGF could be specifically captured with high stability when interacting with covalently immobilized K5 peptide (a synthetic peptide designed to bind to the E5 moiety of chimeric hVEGF). This immobilization strategy was applied to glass substrates and chimeric hVEGF was shown to be maintained in a functionally active state following capture. Altogether, our data demonstrated that stable hVEGF capture can be performed via coiled-coil interactions without impacting hVEGF bioactivity, thus opening up the way to future applications in the field of tissue engineering and regenerative medicine.


Assuntos
Proteínas de Fluorescência Verde/química , Engenharia de Proteínas/métodos , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento/ultraestrutura , Proteínas Recombinantes de Fusão/síntese química , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/ultraestrutura , Sítios de Ligação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/ultraestrutura , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/ultraestrutura , Fator A de Crescimento do Endotélio Vascular/genética
13.
BMC Cell Biol ; 13: 18, 2012 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-22762146

RESUMO

BACKGROUND: The tumor microenvironment contains a vast array of pro- and anti-inflammatory cytokines that alter myelopoiesis and lead to the maturation of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). Incubating bone marrow (BM) precursors with a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) generated a tumor-infiltrating MDSC-like population that impaired anti-tumor specific T-cell functions. This in vitro experimental approach was used to simulate MDSC maturation, and the cellular metabolic response was then monitored. A complementary experimental model that inhibited L-arginine (L-Arg) metabolizing enzymes in MSC-1 cells, an immortalized cell line derived from primary MDSCs, was used to study the metabolic events related to immunosuppression. RESULTS: Exposure of BM cells to GM-CSF and IL-6 activated, within 24 h, L-Arg metabolizing enzymes which are responsible for the MDSCs immunosuppressive potential. This was accompanied by an increased uptake of L-glutamine (L-Gln) and glucose, the latter being metabolized by anaerobic glycolysis. The up-regulation of nutrient uptake lead to the accumulation of TCA cycle intermediates and lactate as well as the endogenous synthesis of L-Arg and the production of energy-rich nucleotides. Moreover, inhibition of L-Arg metabolism in MSC-1 cells down-regulated central carbon metabolism activity, including glycolysis, glutaminolysis and TCA cycle activity, and led to a deterioration of cell bioenergetic status. The simultaneous increase of cell specific concentrations of ATP and a decrease in ATP-to-ADP ratio in BM-derived MDSCs suggested cells were metabolically active during maturation. Moreover, AMP-activated protein kinase (AMPK) was activated during MDSC maturation in GM-CSF and IL-6-treated cultures, as revealed by the continuous increase of AMP-to-ATP ratios and the phosphorylation of AMPK. Likewise, AMPK activity was decreased in MSC-1 cells when L-Arg metabolizing enzymes were inhibited. Finally, inhibition of AMPK activity by the specific inhibitor Compound C (Comp-C) resulted in the inhibition of L-Arg metabolizing enzyme activity and abolished MDSCs immunosuppressive activity. CONCLUSIONS: We anticipate that the inhibition of AMPK and the control of metabolic fluxes may be considered as a novel therapeutic target for the recovery of the immunosurveillance process in cancer-bearing hosts.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-6/farmacologia , Células Mieloides/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Arginina/metabolismo , Células da Medula Óssea/citologia , Células Cultivadas , Glucose/metabolismo , Glutamina/metabolismo , Glicólise , Humanos , Imunossupressão , Células Mieloides/citologia , Células Mieloides/metabolismo
14.
J Immunol Methods ; 362(1-2): 161-7, 2010 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-20869967

RESUMO

The de novo designed heterodimeric E/K coiled-coil system has been previously demonstrated to be an excellent capture/dimerization system applicable to various needs in both biotechnology and pharmaceutical fields. Those include controlled protein dimerization, capture, purification and Western-blot detection. We here report the development of a new generation of ELISA test based on coiled-coil interactions for the direct quantitation of coil-tagged epidermal growth factor (EGF). The new approach was evaluated for its specificity, plate storability and reusability as well as for convenience when compared to commercially available systems. Our results show a similar affinity/sensitivity to standard capturing antibody-based ELISA systems and an improved affinity/sensitivity when compared to the commercially available Ni-NTA capture system. The E/K coiled-coil ELISA system was validated with respect to recovery, intra- and inter-assay variations. The practical working range was estimated to be between 5.2 and 34,000 pM. Furthermore, the storability and reusability of the plates was greater than the two aforementioned systems, suggesting that the E/K coiled-coil system is a good alternative to traditional tags such as poly-histidine for the development of ELISA tests aiming at quantitating coil-tagged proteins.


Assuntos
Fator de Crescimento Epidérmico/análise , Ensaio de Imunoadsorção Enzimática/métodos , Fator de Crescimento Epidérmico/imunologia , Humanos , Estrutura Secundária de Proteína , Sensibilidade e Especificidade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA