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.