부산대학교 도서관

Preparations of recombinant human vascular endothelial growth factor (VEGF165) expressed in Chinese hamster ovary (CHO) cells andEscherichia coliwere compared using a variety of analytical methods. Amino terminal sequence analyses of both the CHO- andE. coli-derived VEGF165confirmed the predicted amino terminal sequence for VEGF165, although the CHO VEGF165exhibited a heterogeneous amino terminus with sequences beginning at Ala-1 (76%), Pro-2 (4%), Ala-4 (13%), and Glu-5 (7%). Tryptic digests of reduced and carboxymethylated CHO- andE. coli-derived VEGF165were examined by LC/MS analyses, indicating equivalent primary structure, except for the glycosylation at Asn-75 in the CHO-derived VEGF165. The N-linked carbohydrate in the CHO-derived VEGF165was determined to be a complex fucosylated biantennary structure. The data obtained from LC/MS analysis and amino terminal sequence analysis of VEGF165confirmed 98% of the primary structure. Disulfide linkages for the eight cysteine residues in the carboxyl terminal heparin binding domain were assigned by amino terminal sequencing of fragments produced by tryptic digests of each native molecule. The following disulfides have been identified for both CHO- andE. coli-derived VEGF165: Cys-117 and Cys-135, Cys-120 and Cys-137, Cys-139 and Cys-158, plus Cys-146 and Cys-160. Plasmin cleavage of VEGF165yields an N-terminal homodimeric VEGF110and a 55-amino-acid carboxyl terminal domain. VEGF110was resistant to further proteolytic or chemical digestion such that the disulfide linkages were not elucidated. The 55-amino-acid carboxyl terminal region of VEGF165appears to be a unique heparin binding domain with no known protein homology.