Butyl toyopearl 650 m

All chemicals used in this study were of the highest grade available and were obtained from Sinopharm Chemical Reagent (Nanjing, China). Toyopearl DEAE-650 M and butyl-Toyopearl 650 M were obtained from Tosoh (Osaka, Japan). Chitopearl BCW-2605 and Chitopearl BCW-2503 (chitosan beads) were purchased from Fujibo (Tokyo, Japan).

G-rhChM-I was expressed in Chinese hamster ovary (CHO) cells and purified from culture supernatants as described previously [6] (link). NG-rhChM-I was expressed in E. coli BL21 and purified as described [13] (link). The disulfide pairings were confirmed by trypsin digestion and peptide mapping of disulfide-bridged G-rhChM-I and NG-rhChM-I [6] (link), [13] (link). The N-terminal deleted 14-kDa rhChM-I (ΔN-rhChM-I) was prepared by incubation of NG-rhChM-I with V8 protease (Pierce, Rockford, IL), and the resultant products were then purified by Butyl-Toyopearl 650 M (Tosoh, Tokyo, Japan) and reversed-phase HPLC [13] (link). Recombinant human vascular endothelial growth factor-A₁₆₅ (VEGF-A₁₆₅) and human vitronectin were purchased from R&D Systems (Minneapolis, MN) and BD Biosciences (Bedford, MA), respectively. Other chemicals were purchased from Sigma (St Louis, MO).