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 May 5 |
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Keywords
- Antiangiogenic polypeptide
- Apolipoprotein A
- Crystal
- Formulation
- Kringle
ASJC Scopus subject areas
- Bioengineering
- Biotechnology
- Biochemistry
- Biochemistry, Genetics and Molecular Biology(all)
- Chemistry (miscellaneous)
- Applied Microbiology and Biotechnology
- Food Science
Cite this
Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A : Crystallization applied to purification and formulation. / Kang, Kwan Yub; Park, Jung Hwan; Lim, In Hwan; Kim, Sung Geun; Park, See Hyoung; Kim, Won Kyung; Hong, Jeong Woon; You, Hyun Kyung; Jung, Kyung Hwan; Kim, Chan Wha.
In: Bioscience, Biotechnology and Biochemistry, Vol. 70, No. 4, 05.05.2006, p. 916-925.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A
T2 - Crystallization applied to purification and formulation
AU - Kang, Kwan Yub
AU - Park, Jung Hwan
AU - Lim, In Hwan
AU - Kim, Sung Geun
AU - Park, See Hyoung
AU - Kim, Won Kyung
AU - Hong, Jeong Woon
AU - You, Hyun Kyung
AU - Jung, Kyung Hwan
AU - Kim, Chan Wha
PY - 2006/5/5
Y1 - 2006/5/5
N2 - 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.
AB - 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.
KW - Antiangiogenic polypeptide
KW - Apolipoprotein A
KW - Crystal
KW - Formulation
KW - Kringle
UR - http://www.scopus.com/inward/record.url?scp=33646142525&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33646142525&partnerID=8YFLogxK
U2 - 10.1271/bbb.70.916
DO - 10.1271/bbb.70.916
M3 - Article
C2 - 16636459
AN - SCOPUS:33646142525
VL - 70
SP - 916
EP - 925
JO - Bioscience, Biotechnology and Biochemistry
JF - Bioscience, Biotechnology and Biochemistry
SN - 0916-8451
IS - 4
ER -