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.
Original language | English |
---|---|
Pages (from-to) | 916-925 |
Number of pages | 10 |
Journal | Bioscience, Biotechnology and Biochemistry |
Volume | 70 |
Issue number | 4 |
DOIs | |
Publication status | Published - 2006 |
Keywords
- Antiangiogenic polypeptide
- Apolipoprotein A
- Crystal
- Formulation
- Kringle
ASJC Scopus subject areas
- Biotechnology
- Analytical Chemistry
- Biochemistry
- Applied Microbiology and Biotechnology
- Molecular Biology
- Organic Chemistry