쌍기능성 폴리에틸렌 글리콜을 이용한 rhEGF의 이량체 PEGylation

Abstract

EGF(Epidermal growth factor)는 상피세포 성장인자로서 표피세포의 분열증식 속도를 조절하여 세포의 증식을 촉진시켜 상처치료에 이용되고 있다. EGF가 EGF receptor와 binding하여 이량체를 만들고 receptor tyrosine kinase를 활성 시킨다. 하지만 이 과정은 EGF가 2-3분 이내에 EGF receptor와 반응하지 못하면 ubiquitination되어 사라지게 되므로 EGF와 EGF receptor가 좀 더 효과적으로 반응할 수 있어야 한다. 본 연구에서는 기존의 methoxy-PEG (polyethylene glycol)를 이용하여 PEGylation하는 방법과 달리 bi-functional PEG aldehyde (3.4 kDa)를 이용하여 하나의 PEG에 두 개의 EGF를 접합시켜 아령형 PEGylated EGF를 생산하였다. EGF의 amine기와 PEG의 aldehyde를 reductive amination 반응을 통해 접합시켰고, GPC (Gel Permeation Chromatography)와 IEX (Ion Exchange Chromatography)의 두 단계 크로마토그래피 공정을 거쳐 di-EGF PEGylate를 분리하였다. MALDI -TOF (Matrix Assisted Laser Desorption-Time of flight) mass spectrometry에 의한 질량 분석과 Reversed Phase-HPLC를 통해 di-EGF PEGylate의 생성과 순도를 확인하였다. Di-EGF PEGylate의 생물학적 활성은 mitogenic assay를 이용하여 측정한 결과 native EGF에 비해 생물학적 활성은 약 20% 이하 수준으로 감소하였으나 DSC (Differential Scanning Calorimetry)를 통해 측정한 온도 안정성은 native EGF 보다 Tm 값이 27℃가량 향상되었다.; Recombinant human epidermal growth factor (rhEGF) is a 53-amino acid polypeptide, which exhibits potent mitogenic activities, such as cell proliferation and differentiation of various tissues and cells. EGF binds to the EGF receptor on cell membrane forming a dimerized complex, which is then internalized within cells. The internalized EGF receptor has a tyrosine kinase activity, thus initiating a series of signal transduction cascades. It has been demonstrated that the site of PEGylation was very important and that it had major effects on their biologic activities. In this study, We PEGylated two EGF molecules to a PEG chain, i. e., di-EGF PEGylation, to form a dumbbell-like structure. We modified the N-terminus of EGF in a site-specific manner using bi-functional PEG-aldehyde (Mw 3400). We used GPC and IEX chromatography to purify dimeric-EGF PEGylates. Various characterization methods including MALDI-TOF and RP-HPLC techniques were used for separation of the PEGylates and identification of the dimeric-EGF PEGylates. And the site of PEGylation was identified through tryptic peptide mapping and MALDI-TOF techniques. 2-DE was performed and the result were there was no change in isoelectric point of the dimeric-EGF PEGylate. ITC experiment was performed to determine the limiting species between PEG and EGF, by altering the titrating sequence. The result seemed to suggest PEG was limiting the PEGylation reaction. The di-EGF PEGylate showed significantly reduced cell proliferating activity determined by mitogenic assay. Nevertheless, the thermal stability was improved.