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세포질에서 발현된 항-KIFC1재조합 항체의 구조 및 기능적 특성
- Alternative Title
- Youngsil Seo
- Author(s)
- 서영실
- Alternative Author(s)
- Youngsil Seo
- Advisor
- 권명희
- Department
- 일반대학원 의생명과학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2019-02
- Language
- eng
- Alternative Abstract
- Intrabodies, antibodies expressed in cells, are widely used in the life science field to regulate the function of intracellular antigens. Most of studies about intrabodies expressed in the cytosol have been performed on single-chain variable fragment (scFv) or single domain antibodies containing few disulfide bonds and simple structure. Because it is known that the disulfide bond is hardly formed in the reducing condition of the cytosol, the antigen-binding site may not be properly formed when the complex structure of immunoglobulin G (IgG) is expressed in the cytosol. In this study, IgG was expressed in the cytosol for the first time to increase the utilization of cytosolically expressed antibody. First, in order to confirm the effect of the variable region on the activity of the cytosolic IgG intrabodies, three anti-KIFC1 (kinesin family C1) IgGs and one anti-nucleic acid IgG that consist of the same human-derived constant regions and different mouse-derived variable regions were expressed in the cytosol. Analysis of the cytosolic IgG intrabodies revealed that the antigen-binding activity and the association of heavy and light chains were different according to the variable region sequence. In order to investigate the effect of the constant region folding on the characteristics of the cytosolic IgG intrabodies, cysteine residues in the constant regions were replaced with serine residues to inhibit formation of inter- and intra-chain disulfide bonds. The results suggested that integrity of the constant region affects formation of antigen-binding site of the variable region. It was also observed that the heavy and light chains were not associated in the absence of intra-chain disulfide bond of constant region. The presence of the disulfide bonds in cytosolic IgG intrabody was confirmed by direct PEGylation of cysteine residues. We also compared the re-folding of fully reduced ER-directed IgG and cytosolic IgG intrabody. It was confirmed that even if the protein had completely the same amino acid sequence, it was folded differently depending on the location of expression. In the case of scFv form of cytosolic 6C407 and 2C281, they retained its antigen-binding activity in the scFv form as well as IgG form. Further, cytosolic 6C407 scFv expression increased multipolar spindle formation and prolonged mitotic duration, suggesting that 6C407 scFv inhibits centrosome clustering function of KIFC1. This study is expected to contribute to the understanding of the structural and functional properties of cytosolically expressed antibodies.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Biomedical Sciences > 4. Theses(Ph.D)
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