서지주요정보
당전이 효소가 도입된 CHO 세포에서 생산된 재조합 EPO의 당쇄구조 분석 = Glycan analysis of recombinant erythropoietin produced by glycosyltransferase-engineered Chinese hamster ovary cells
서명 / 저자 당전이 효소가 도입된 CHO 세포에서 생산된 재조합 EPO의 당쇄구조 분석 = Glycan analysis of recombinant erythropoietin produced by glycosyltransferase-engineered Chinese hamster ovary cells / 임혜림.
저자명 임혜림 ; Lim, Hye-Rim
발행사항 [대전 : 한국과학기술원, 2005].
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등록번호

8015935

소장위치/청구기호

학술문화관(문화관) 보존서고

MBS 05017

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초록정보

The attachment of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. Galactosyltransferase (GT) and sialyltransferase (ST) are responsible for the terminal galactosylation and sialylation, respectively. In order to increase the sialylation of a protein, human α2,3-ST and β1,4-GT were engineered into Chinese hamster ovary (CHO) cells which produce recombinant human erythropoetin (EPO). Recombinant human EPO was purified from the culture supernatant by an immunoaffinity chromatography and N-glycans were released from the purified EPO, derivatized with 2-aminopyridine, and the relative sialylation of EPO was structually evaluated by DEAE chromatography and 2-D HPLC(ODS and Amide-80). When both α2,3-ST andβ1,4-GT were expressed in CHO cells (GTST15), more sialylated glycans were produced than those of control (EC1). In detail, relative amounts of di- and tri-sialylated glycans were increased while those of neutral and mono-sialylated glycans were decreased. Specially, tri-sialylated glycans were remarkably increased. Tri-sialylated glycans from EPO in GTST15 cells were isolated, and micro-structures of glycans were elucidated by 2-D HPLC. Most abundant glycans were tetra-antennary structures. In a case of GTST15 cells, the relative protion of tetra-antennary glycans with 3 galactose(Gal) residues was decreased compared to that of control(EC1) cells. Also, tri-antennary glycans with 3 lactosamine(Gal-NeuNAc) units and tetra-antennary glycans with 3 Gal residues and 5 lactosamine units were newly generated in GTST15 cells. The coexpression of the α2,3-ST andβ1,4-GT, however did not affect to the cell growth and EPO productivity of CHO cells. Expression of α2,3-ST andβ1,4-GT may be beneficial in CHO cells for producing a pharmaceutical glycosylation.

1. CHO 세포에 당전이 효소인 β1,4-Galactosyltransferase(GT)와 α 2,3-sialyltransferase(ST)를 도입하였다. 2. CHO 세포배양을 통해 얻어진 배양액에서 Immunoaffinity column을 이용하여 EPO를 순수분리정제 하였다. 3. GT와 ST를 도입하여 얻어진 EPO(GTST15)의 경우 control(EC1)에 비해 tri-ialylated glycan 양이 EC1에서의 17.3%에서 35.5%까지 향상이 되었고 GTST15에서의 mono-sialylated glycan의 양은 EC1에서의 31.2%에서 18.1%까지 줄어든 것을 볼 수 있었다. 4. 2-D HPLC를 이용한 당쇄구조 분석에서 EC1에서 상대적인 양을 가장 많이 차지하는 Gal가 3개, lactosamine unit3개, Fuc를 갖는 tetra-antennay 구조는 GTST15에서 상대적인 양이 많이 줄어든 것을 볼 수 있었다. 반면 Gal 4개, lactosamine unit5개, Fuc를 갖는 tetra-antennary 구조는 GTST에서 현저하게 증가된 것을 볼 수 있었다.

서지기타정보

서지기타정보
청구기호 {MBS 05017
형태사항 vii, 60 p. : 삽도 ; 26 cm
언어 한국어
일반주기 저자명의 영문표기 : Hye-Rim Lim
지도교수의 한글표기 : 김정회
지도교수의 영문표기 : Jung-Hoe Kim
학위논문 학위논문(석사) - 한국과학기술원 : 생명과학과,
서지주기 참고문헌 : p. 43-49
주제 차이니즈 햄스터 오버리 세포
에리쓰로포에틴
갈락토오스 전이효소
시알산 전이효
DEAE 크로마토그래피
CHO
erythropoietin
sialylation
galactosylation
galactosyltransferase
sialyltransferase
DEAE Chromatography
2-D HPLCnjugation
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