Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A: Crystallization applied to purification and formulation

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2006, Vol.70 (4), p.916-925
• Main Authors: Kang, K.Y.(Korea Univ., Seoul (Korea R.)), Park, J.H, Lim, I.H, Kim, S.G, Park, S.H, Kim, W.K, Hong, J.W, You, H.K, Jung, K.H, Kim, C.W
• Format: Article
• Language: English
• Subjects: Angiogenesis Inhibitors - chemistry; Angiogenesis Inhibitors - isolation & purification; Angiogenesis Inhibitors - metabolism; Angiogenesis Inhibitors - pharmacology; antiangiogenic polypeptide; apolipoprotein A; Apolipoproteins A - chemistry; Apolipoproteins A - isolation & purification; Apolipoproteins A - metabolism; Apolipoproteins A - pharmacology; APOPROTEINAS; APOPROTEINE; APOPROTEINS; Biological and medical sciences; Cell Movement - drug effects; Cells, Cultured; Chromatography, Liquid; CRISTALIZACION; CRISTALLISATION; crystal; CRYSTALLIZATION; Endothelial Cells - cytology; Endothelial Cells - drug effects; FORMULACIONES; FORMULATION; FORMULATIONS; Fundamental and applied biological sciences. Psychology; GENERO HUMANO; GENRE HUMAIN; Humans; Hydrogen-Ion Concentration; kringle; Kringles; MANKIND; Mass Spectrometry; Molecular Weight; PEPTIDE; PEPTIDES; PEPTIDOS; PURIFICACION; PURIFICATION; Solubility; Umbilical Cord - cytology; Umbilical Cord - drug effects
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.70.916

In this study, the Kringle V domain (Glusup(4225)-Sersup(4310)) 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.