세포질에서 발현된 항-KIFC1재조합 항체의 구조 및 기능적 특성
Structural and Functional Properties of the Recombinant anti-KIFC1 Antibodies Expressed in the Cytosol
- 주제(키워드) Intrabody , IgG , scFv , Cytosol expression , Disulfide bond
- 발행기관 아주대학교
- 지도교수 권명희
- 발행년도 2019
- 학위수여년월 2019. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028641
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
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.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 11
A. Plasmids 11
B. Purification of proteins 14
C. Preparation of cell lysates 15
D. Enzyme-linked immunosorbent assay (ELISA) 16
E. Confocal microscopy 19
F. Immunoblotting 21
G. Immunoprecipitation (IP) 22
H. Measurement of antigen-binding affinity by octet 22
I. Analysis of disulfide bonds by Cys-residue PEGylation 23
J. Analysis of assembly dynamics 24
K. Fluorescence microscopy 24
L. Live cell imaging 25
M. Analysis of meta-to-anaphase delay 25
III. RESULTS 27
A. Expression of IgGs in the cytosol 27
B. Antigen-binding activity of cytosolic IgGs 29
C. Association of heavy and light chains of cytosolic IgGs 33
D. H:L association in the absence of formation of the correct antigen-binding site 36
E. Effect of structural integrity of the constant region on H:L association and formation of the correct antigen-binding site in cytosolic IgGs 39
F. Importance of intra-chain disulfide bonds in H:L association and antigen-binding site formation of cytosolic IgGs 42
G. Partial formation of intra-chain disulfide bonds in heavy and light chains of cytosolic IgGs 44
H. Differences in assembly of IgG1s expressed in the cytosol or via the ER 48
I. Antigen-binding affinity of purified chimeric anti-KIFC1 IgGs 50
J. Antigen-binding activity of purified anti-KIFC1 scFvs 57
K. Antigen-binding activity of cytosolic scFvs 59
L. Increased multipolar spindle in anti-KIFC1 scFv expressing cells 63
IV. DISCUSSION 69
V. CONCLUSION 73
REFERENCES 74
국문요약 83
