Crystallization of Antiangiogenic Kringle V Derived from Human Apolipoprotein A : Crystallization Applied to Purification and Formulation
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In this study, the Kringle V domain (Glu<SUP>4225</SUP>-Ser<SUP>4310</SUP>) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in <I>Pichia pastoris</I>, and was purified <I>via</I> 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.
- Agricultural and Biological Chemistry
Agricultural and Biological Chemistry 70(4), 916-925, 2006-04-23
Japan Society for Bioscience, Biotechnology, and Agrochemistry