Expression of the Amino-Terminal Domain of Platelet Glycoprotein Ibα: Exploitation of a Calmodulin Tag for Determination of Its Functional Activity

Platelet glycoprotein (GP) Ibα is a component of the GPIb-IX receptor complex, which is involved in multiple physiological and pathological processes, including platelet adhesion at sites of vascular injury, thrombin binding, Bernard-Soulier syndrome, platelet-type von Willebrand disease, and immune...

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Published in:Protein expression and purification 2001-07, Vol.22 (2), p.200-210
Main Authors: Li, Chester Q, Ye, Pei, Cao, Zhao-feng, Wang, Huifang, Lu, Lan, Nicastro, Peter, Wood, Emma, Robert, Justin J, Ouwehand, Willem H, Hill, Fergal, López, José A, Wardell, Mark R
Format: Article
Language:English
Subjects:
Animals
Baculoviridae - genetics
Calmodulin - genetics
Calmodulin - physiology
Genetic Vectors
Humans
Peptide Fragments - biosynthesis
Peptide Fragments - genetics
Peptide Fragments - metabolism
Peptide Fragments - physiology
Platelet Glycoprotein GPIb-IX Complex - biosynthesis
Platelet Glycoprotein GPIb-IX Complex - genetics
Platelet Glycoprotein GPIb-IX Complex - metabolism
Platelet Glycoprotein GPIb-IX Complex - physiology
Protein Binding - genetics
Protein Processing, Post-Translational - genetics
Protein Structure, Tertiary - genetics
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - metabolism
Spodoptera - genetics
Sulfates - metabolism
Thrombin - metabolism
von Willebrand Factor - metabolism
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Summary:Platelet glycoprotein (GP) Ibα is a component of the GPIb-IX receptor complex, which is involved in multiple physiological and pathological processes, including platelet adhesion at sites of vascular injury, thrombin binding, Bernard-Soulier syndrome, platelet-type von Willebrand disease, and immune-mediated thrombocytopenias. The amino-terminal domain of ∼300 residues of GPIbα mediates both normal biological function (by providing the sites for direct ligand interaction) and aberrant function (through amino acid substitutions). To investigate the molecular interactions mediated by this region of GPIbα, we have developed a recombinant baculovirus to facilitate its expression as a calmodulin fusion protein from insect cells. By employing the calmodulin tag, the fusion protein could be obtained at >90% purity after a single isolation step at yields of 8 mg/L of insect cell medium (purified fusion protein). The recombinant GPIbα fragment was shown to be posttranslationally sulfated and glycosylated, although its glycosylation differed from that of the equivalent GPIbα fragment isolated from human platelets. The differential glycosylation, however, did not affect the function of the recombinant GPIbα fragment in either von Willebrand factor (vWf) or thrombin binding as these were both found to be identical to those of the same-length GPIbα fragment derived from human platelets. The calmodulin tag was also exploited in the development of assays to measure directly vWf and thrombin binding, since it did not interfere with either, demonstrating the feasibility for the use of this soluble receptor fusion protein in detailed biophysical assays to investigate the molecular mode of binding of platelet glycoprotein Ibα to these ligands.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.2001.1441