Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop conta...

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Published in:	Protein expression and purification Vol. 31; no. 2; pp. 286 - 291
Main Authors:	Butera, Diego, Skielka, Karen, Ann McLane, Mary, Paquette-Straub, Carrie, Ducancel, Frédéric, Maria Moura da Silva, Ana
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-10-2003
Subjects:	Alkaline Phosphatase - analysis Alkaline Phosphatase - genetics Alkaline Phosphatase - metabolism Animals Blood Platelets - metabolism Cloning, Molecular Disintegrin Disintegrins - analysis Disintegrins - genetics Disintegrins - metabolism Dose-Response Relationship, Drug Eristostatin Genetic Vectors Humans Molecular marker Peptides - analysis Peptides - genetics Peptides - metabolism Platelet Aggregation Inhibitors - analysis Platelet Aggregation Inhibitors - metabolism Protein Binding Recombinant Fusion Proteins - analysis Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Viper Venoms - analysis Viper Venoms - genetics Viper Venoms - metabolism α IIbβ 3 integrin α IIbβ 3 integrin Disintegrin Eristostatin Molecular marker
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Summary:	Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to α IIbβ 3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS–PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an α IIbβ 3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and α IIbβ 3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the α IIbβ 3 integrin is involved.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1046-5928 1096-0279
DOI:	10.1016/S1046-5928(03)00169-4