Expression of the amino-terminal domain of platelet glycoprotein Ib alpha: exploitation of a calmodulin tag for determination of its functional activity.

Abstract

Platelet glycoprotein (GP) Ibalpha 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 approximately 300 residues of GPIbalpha 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 GPIbalpha, 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 GPIbalpha fragment was shown to be posttranslationally sulfated and glycosylated, although its glycosylation differed from that of the equivalent GPIbalpha fragment isolated from human platelets. The differential glycosylation, however, did not affect the function of the recombinant GPIbalpha fragment in either von Willebrand factor (vWf) or thrombin binding as these were both found to be identical to those of the same-length GPIbalpha 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 Ibalpha to these ligands.

• Authors: C Q Li, P Ye, Cao Zf, H Wang, L Lu, P Nicastro, E Wood, J J Robert, W H Ouwehand, F Hill, J A López, M R Wardell
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 22 Issue 2 Pg. 200-10 (Jul 2001) ISSN: 1046-5928 [Print] United States
• PMID: 11437595 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
• Copyright: Copyright 2001 Academic Press.

Chemical References

• Calmodulin
• Peptide Fragments
• Platelet Glycoprotein GPIb-IX Complex
• Recombinant Fusion Proteins
• Sulfates
• glycocalicin
• von Willebrand Factor
• Thrombin

Topics

• Animals
• Baculoviridae (genetics)
• Calmodulin (genetics, physiology)
• Genetic Vectors
• Humans
• Peptide Fragments (biosynthesis, genetics, metabolism, physiology)
• Platelet Glycoprotein GPIb-IX Complex (biosynthesis, genetics, metabolism, physiology)
• Protein Binding (genetics)
• Protein Processing, Post-Translational (genetics)
• Protein Structure, Tertiary (genetics)
• Recombinant Fusion Proteins (biosynthesis, metabolism)
• Spodoptera (genetics)
• Sulfates (metabolism)
• Thrombin (metabolism)
• von Willebrand Factor (metabolism)