Tie-2, also known as Tek, is a 145 kD, type I transmembrane glycoprotein receptor tyrosine kinase that is a receptor for angiopoietins. 1 The 1120 amino acid (aa) rat Tie-2 precursor contains an 18 aa signal sequence, a 723 aa extracellular domain (ECD), a 25 aa transmembrane segment, and a 354 aa cytoplasmic tail. 2 The ECD contains two C2 Ig-like domains, three EGF-like motifs, and three fibronectin type III repeats. The cytoplasmic region has a split tyrosine kinase domain and presumably autophosphorylates as a ligand-induced homodimer. 3 Rat Tie-2 ECD shares 96%, 90% and 89% aa identity with mouse, human and bovine Tie-2, respectively, and 47% aa identity with rat Tie-1 ECD. Cells known to express Tie-2 include embryonic and adult endothelial cells, hematopoietic stem cells and a circulating population of proangiogenic Tie-2 expressing monocytes (TEM). 4-7 A soluble form of Tie-2, most likely the result of proteolytic cleavage, is found in serum. 8 The four angiopoietins are ligands of Tie-2. Ang-1 and Ang-4 are Tie-2 activators, while Ang-2 and Ang-3 can be activators or inhibitors, depending on context. 1, 9 Tie-2 is said to be important for maintaining vascular integrity. It mediates endothelial cell-smooth muscle cell communication, and inhibits endothelial cell apoptosis, thus maintaining endothelial cell survival. 10-12 It is also absolutely required for embryonic development of the endocardium. 3, 10, 13 While not essential for embryonic hematopoiesis, Ang-1 production by osteoblasts promotes quiescence of Tie-2-expressing bone marrow stem cells. This quiescence is critical for maintaining an ongoing hematopoietic capability. 12, 14, 15
Source: Human CD33 signal peptide (Met 1-Ala 16), Rat Tie-2 (Ala 60-Leu 780), IEGRMD, Human IgG1 (Pro 100-Lys 330); A DNA sequence encoding the signal peptide from human CD33 joined with the extracellular domain of rat Tie-2 (Ala 60-Leu 780; Accession # XP_342864) was fused to the Fc region of human IgG1 via a peptide linker. The chimeric protein was expressed in a mouse myeloma cell line, NS0.
Molecular Mass: The recombinant mature rat Tie-2/Fc chimera, generated by proteolytic removal of the signal peptide, is a disulfide-linked homodimer. Based on N-terminal amino acid sequencing, the recombinant rat Tie-2 protein starts at Ala 60. The predicted molecular mass of the monomer is approximately 107.3 kD. As a result of glycosylation, the recombinant protein migrates as an approximately 120-140 kD protein in SDS-PAGE under reducing conditions. A small amount of free Fc and free rat Tie-2 may be present in the preparation.
Endotoxin Level: < 1.0 EU per 1 μg of the protein as determined by the LAL method.
Activity: Measured in a functional ELISA assay. When rrTIE-2/Fc is immobilized at 4μg/mL (100 μL/well), it binds rhAngiopoietin-2 with a linear range of 0.8-50 ng/mL.
Reconstitution: It is recommended that sterile PBS be added to the vial to prepare a working stock solution of no less than 100ug/ml. The carrier-free protein should be used immediately upon reconstitution to avoid losses in activity due to non-specific binding to the inside surface of the vial. For long term storage as a dilute solution, a carrier protein (e.g. 0.1% HSA or BSA) should be added to the vial.
Storage and Stability: Lyophilized samples are stable for up to twelve months from date of receipt at -20°C. Upon reconstitution, this protein, in the presence of a carrier protein, can be stored under sterile conditions at 2°-8° C for one month or at -20°C in a manual defrost freezer for three months without detectable loss of activity. Avoid repeated freeze-thaw cycles.
