Purification of recombinant Tie1-751
Tie1-751 was subcloned into pcDNA3.1 as described above with a Myc-His tag fused at the C-terminus of the proteins (Tie1-751(6His)). To construct Tie1-751-Fc, the Fc fragment of human IgG1 (from Pro100 to Lys330) was PCR amplified and fused inframe to the 3' end of Tie1-751 in the pcDNA 3.1 vector via restriction digestion using the XhoI-AgeI site. Tie1-751(6His) and Tie1-751-Fc were transiently expressed in HEK 293 cells. Conditioned media were collected 72 hours later. Tie1-751(6His) was purified using a Ni-Sepharose 6 Fast Flow column (GE-Amersham, Piscataway, NJ, USA) and Tie1-751-Fc was purified using a Protein-A Sepharose column (GE-Amersham), following the manufacturer's instructions. Purity of the recombinant proteins was >95% as determined by SDS-PAGE and Coomassie Blue staining.