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황 개질된 재조합 상피성장인자의 위치 특이적 페길레이션 및 특성화

Title
황 개질된 재조합 상피성장인자의 위치 특이적 페길레이션 및 특성화
Other Titles
Site specific PEGylation and Characterization of thiolated rhEGF
Author
김형진
Alternative Author(s)
Kim, Hyung Jin
Advisor(s)
이은규
Issue Date
2009-02
Publisher
한양대학교
Degree
Master
Abstract
EGF(Epidermal growth factor)는 상피세포 성장인자로서 표피세포의 분열증식 속도를 조절하여 세포의 증식을 촉진시켜 상처치료에 이용되고 있다. EGF는 PEG 분자와 공유결합을 이루어 혈액내 안정성을 증가시킨다. 기존의 EGF 표면의 아민기를 대상으로 한 PEGylation 공정은 PEGylation 수율이 높지 않다는 문제점을 가지고 있다. 본 연구에서는 아민기보다 높은 반응성을 가지는 thiol group을 이용하여 PEGylation 수율을 증가시키고자, iminothiolane으로 rhEGF를 thiolation시킨 후 PEG-SH 혹은 PEG-maleimide를 conjugation시켰다. 이를 GPC (Gel Permeation Chromatography)를 통해 분리하고 MALDI -TOF (Matrix Assisted Laser Desorption-Time of flight) mass spectrometry에 의한 질량 분석과 reversed phase-HPLC를 통해 thiolated rhEGF PEGylates의 생성과 순도를 확인하였다. PEG-SH를 사용한 경우에는 직접적인 disulfide 형성을 위한 낮은 반응성 때문에 자체 응집 현상이 촉진되었으나, PEG-maleimide를 사용한 경우에는 thiol기와 thioether 형성 반응에 의해 PEGylation되므로 자체 응집도 거의 발생하지 않고 PEGylation 수율이 95% 이상이었으며, 대부분 (약 86%)이 mono-PEGylates였다.
Covalent conjugation of poly(ethylene glycol) to therapeutic proteins increases their in vivo stability by protecting the protein from degradation, masking its immunogenic sites and reducing clearance. In some case of PEGylation, target protein could have problem which is difficult to conjugate to lysine residue or N-terminus with PEG-aldehyde. To solve this problem, thiolation of target protein was performed before PEGylation with PEG-SH or PEG-maleimide. Epidermal growth factor (EGF) has been used as a medical treatment, since it controls and accelerates the epidermal cell's proliferation. In this study, we demonstrated the PEGylation of the thiolated EGF using 2·iminothiolane·HCl, which can improve the binding interaction between a PEG molecule and a EGF protein and yield of PEGylation. PEGylation was identified through Matrix Assisted Laser Desorption-Time of Flight (MALDI-TOF) techniques, which shows mass analysis and reversed phase-HPLC, which can confirm the existence of tri-, di-, mono-EGF PEGylates and their purity levels. We obtained max. 96% of PEGylation yield which is much higher than previouse PEGylation yield, ca. 60% obtained from PEG-aldehyde. The thiolated EGF-PEGylates have maintained immuno-activity compared to the native EGF by using a Western blotting method. The stability against tempareture and proteinase also determined and showed the greatness of this method. It can apply to 'hard-to-PEGylate' proteins, such as factor ⅷ.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/145539http://hanyang.dcollection.net/common/orgView/200000411881
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIONANOTECHNOLOGY(바이오나노학과) > Theses (Master)
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