Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A: crystallization applied to purification and formulation.
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.
Kang
Kwan-Yub
KY
School of Life Sciences and Biotechnology, Korea University, Seoul, Korea.
Park
Jung-Hwan
JH
Lim
In-Hwan
IH
Kim
Sung-Geun
SG
Park
See-Hyoung
SH
Kim
Won-Kyung
WK
Hong
Jeong-Woon
JW
You
Hyun-Kyung
HK
Jung
Kyung-Hwan
KH
Kim
Chan-Wha
CW
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
D020533:Angiogenesis Inhibitors; D001054:Apolipoproteins A