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항체 절편화와 Fc 결합된 표피성장인자(EGF)의 제조 및 특성 분석

Title
항체 절편화와 Fc 결합된 표피성장인자(EGF)의 제조 및 특성 분석
Author
변현경
Advisor(s)
정봉근
Issue Date
2011-02
Publisher
한양대학교
Degree
Master
Abstract
Fc fragment of an antibody, ca. 25 kD MW, is known to enhance stability of a protein, and thus it is often used as a fusion protein partner to increase the stability of pharmaceutical proteins. In this study, we used a commercially available IdeS protease (FabRICATOR��) to cleave the specific site of rabbit immunoglobulin G (IgG) gamma globulin, and the resulting Fc fragment was separated by Protein A bead. A SPR biosensor study indicated that the Fc fragment showed higher immunobinding ability than the intact antibody. Using rhEGF (epidermal growth factor) as a model protein, we attempted direct chemical conjugation of the C-terminus of Fc and N-terminus of EGF. Two methods were tried: (1) EDC/sulfo-NHS treatment of the C-terminus of Fc, and (2) periodate-oxidation of the oligosaccharide domain of the Fc to aldehyde. Both methods did not yield good results. As an alternative, polyethylene glycol (PEG), a GRAS polymer frequently used to extend the in-serum half-life of injectible protein drugs, was used as a conjugation linker. An activated PEG, ButyrALD-PEG-ButyrALD (both ends with aldehyde groups) with average MW of 10 kD, was used to link the N-terminus of Fc and EGF. SDS-PAGE revealed the conjugation (Fc-PEG-EGF fusion protein with ca. 40 kD MW) was successful, and it was recovered by anion exchange chromatography and size exclusion chromatography. In summary, it is concluded that the chemical conjugation between the two proteins, i.e., Fc and EGF, seems to require a linker molecule, and the bifucntional PEG appeared well suited for that purpose.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/140561http://hanyang.dcollection.net/common/orgView/200000417029
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIONANOTECHNOLOGY(바이오나노학과) > Theses (Master)
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