title: Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A: crystallization applied to purification and formulation.
abstract: In this study, the Kringle V domain (Glu4225-Ser4310) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in Pichia pastoris, and was purified via a process consisting of three chromatographic steps. The chromatographically purified kringle V domain contained a C-terminal serine-deleted form and several high-molecular-weight forms, which were suspected to represent glycosylated derivatives. In order to remove these derivatives, we employed a crystallization process. The crystallization of kringle V resulted in an 85% recovery yield, and also resulted in the complete removal of the aforementioned high-molecular-weight forms. However, we were still able to detect a trace of the C-terminal serine-deleted form. The prepared Kringle V crystals were stable within a pH range of 7.0 to 8.0, and were completely dissolved by dilution, which is a crucial factor in the preparation of a highly concentrated formulation. The chromatogram of the crystallized kringle V on reversed-phase HPLC analysis was identical to that observed without crystallization. Also, we noted that the original anti-wound migration activities of the molecule toward human umbilical vein endothelial cells were completely retained.
authors.affiliation: School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.
authors.forename: Kwan-Yub
authors.initials: KY
authors.lastname: Kang
authors.forename: Jung-Hwan
authors.initials: JH
authors.lastname: Park
authors.forename: In-Hwan
authors.initials: IH
authors.lastname: Lim
authors.forename: Sung-Geun
authors.initials: SG
authors.lastname: Kim
authors.forename: See-Hyoung
authors.initials: SH
authors.lastname: Park
authors.forename: Won-Kyung
authors.initials: WK
authors.lastname: Kim
authors.forename: Jeong-Woon
authors.initials: JW
authors.lastname: Hong
authors.forename: Hyun-Kyung
authors.initials: HK
authors.lastname: You
authors.forename: Kyung-Hwan
authors.initials: KH
authors.lastname: Jung
authors.forename: Chan-Wha
authors.initials: CW
authors.lastname: Kim
mesh_terms: D020533:Angiogenesis Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D001054:Apolipoproteins A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D003460:Crystallization; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018082:Kringles; D013058:Mass Spectrometry; D008970:Molecular Weight; D012995:Solubility; D014470:Umbilical Cord / Q000166:cytology / Q000187:drug effects
chemical_list: D020533:Angiogenesis Inhibitors; D001054:Apolipoproteins A