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Expression of Recombinant Human Erythropoietin With Glycosylation Modification In Hek293T Cells

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Stability of erythropoietin (EPO) depends on its glycosylation states. With more glycosylation sites, the EPO protein will be more stable and also increase its half-life. A construct of recombinant human erythropoietin (rhEPO) which contains 2 additional N-link for glycosylation were designed. Based on translation analysis using ORF (open reading frame)-finder and protein alignment analysis using blast-p of NCBI home page, expected recombinant hEPO with additional 6-histidin -taq in carboxyl terminus was expressed. HEK293T cells were transfected with recombinant plasmid containing rhEPO by using calcium phosphate method. Expression of rhEPO was detected by dot blot and Western blot analysis using hEPO anti body as the primary anti body and antirabbit anti body with alkaline phospatase linked as the secondaryantibody. The bands were detected by BCIP/NBT color development substrate. The data indicated detection of EPO in culture medium of transfected HEK293T cells.
Key words: HEK293T cell, calcium phosphate transfection, N-linked glycosylation, recombinant human erythropoietin.

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Detail Information

Series Title

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Call Number

Indonesian Journal of Pharmacy, 23 (3) 2012 : 177-

Publisher

Yogyakarta : Faculty of Pharmacy Universitas Gajah Mada Yogyakarta., 2012

Collation

6p

Language

ISBN/ISSN

0126-1037

Classification

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Content Type

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Media Type

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Carrier Type

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Edition

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Subject(s)

GLYCOSYLATION
ERYTHROPOETIN

Specific Detail Info

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