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1.
Biotechnol Appl Biochem ; 45(Pt 3): 167-72, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16872272

RESUMO

Here, we demonstrate for the first time that the hollow-fibre bioreactor is an excellent tool for the production of Drosophila-expressed recombinant proteins. Using the example of the soluble extracellular portion of the human IL-5 (interleukin 5) receptor alpha expression in S2 (Schneider's Drosophila melanogaster cell line 2) cells, we found that it is possible to produce multi-milligram amounts of functional recombinant protein continuously for several months on a laboratory scale with minimal maintenance requirements. The insect cells grow to high density and express concentrated functional recombinant protein in a small volume, simplifying and economizing downstream purification.


Assuntos
Reatores Biológicos , Drosophila melanogaster/citologia , Subunidade alfa de Receptor de Interleucina-5/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Ligação Competitiva , Técnicas Biossensoriais , Células Cultivadas , Drosophila melanogaster/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-5/genética , Subunidade alfa de Receptor de Interleucina-5/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície
2.
Biochemistry ; 45(4): 1106-15, 2006 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-16430207

RESUMO

The cyclic peptide AF17121 (VDECWRIIASHTWFCAEE) is a library-derived antagonist for human Interleukin-5 receptor alpha (IL5Ralpha). We have previously demonstrated that AF17121 mimics Interleukin-5 (IL5) by binding in a region of IL5Ralpha that overlaps the IL5 binding epitope. In the present study, to explore the functional importance of the amino acid residues of AF17121 required for effective binding to, and antagonism of, IL5Ralpha, each charged residue was subjected to site-directed mutagenesis and examined for IL5Ralpha interaction by using a surface plasmon resonance biosensor. One residue, Arg(6), was found to be essential for receptor antagonism; its replacement with either alanine or lysine completely abolished the interaction between AF17121 and IL5Ralpha. Other charged residues play modulatory roles. One class consists of the N-terminal acidic cluster (Asp(2) and Glu(3)) for which alanine replacement decreased the association rate. A second class consists of His(11) and the C-terminal acidic cluster (Glu(17) and Glu(18)) for which alanine replacement increased the dissociation rate. Binding model analysis of the mutants of the latter class of residues indicated the existence of conformational rearrangement during the interaction. On the basis of these results, we propose a model in which Arg(6) and N-terminal acidic residues drive the encounter complex, while Arg(6), His(11), and C-terminal acidic residues are involved in stabilizing the final complex. These data argue that the charged residues of AF17121 are utilized asymmetrically in the pathway of inhibitor-receptor complex formation to deactivate the receptor function. The results also help focus emerging models for the mechanism by which IL5 activates the IL5Ralpha-betac receptor system.


Assuntos
Peptídeos Cíclicos/metabolismo , Receptores de Interleucina/metabolismo , Sequência de Aminoácidos , Animais , Arginina/química , Arginina/metabolismo , Drosophila/metabolismo , Epitopos/química , Epitopos/metabolismo , Histidina/química , Histidina/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-5 , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina/química , Receptores de Interleucina-5 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Temperatura , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
3.
J Biol Chem ; 280(24): 22951-61, 2005 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-15826943

RESUMO

The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor alpha (IL5Ralpha) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N- and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556-568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5Ralpha interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide.receptor complex is comparable with that of the cytokine.receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5Ralpha, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its binding footprint on IL5Ralpha by alanine substitution of Asp(55), Asp(56), Glu(58), Lys(186), Arg(188), and Arg(297) of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5Ralpha, in particular Asp(55), Arg(188), and Arg(297) in the D1, D2, and D3 domains, respectively. This footprint represents a significant subset of that for IL5 binding. The fact that AF17121 mimics the receptor binding capability of IL5 but antagonizes biological function evokes several models for how IL5 induces activation of the multisubunit receptor system.


Assuntos
Interleucina-5/química , Receptores de Interleucina/química , Alanina/química , Sequência de Aminoácidos , Arginina/química , Sequência de Bases , Ligação Competitiva , Técnicas Biossensoriais , Linhagem Celular Tumoral , Citocinas/química , DNA/química , Epitopos/química , Escherichia coli/metabolismo , Fibronectinas/química , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Humanos , Concentração Inibidora 50 , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Subunidade alfa de Receptor de Interleucina-5 , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Plasmídeos/metabolismo , Polímeros/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Tiorredoxinas/química , Fatores de Tempo
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