Cloning and expression of biological active human Erythropoietin in mammalian cells

Abstract

Erythropoietin (EPO) is a hematopoietic growth factor which stimulates erythropoiesis (red blood cell production). One widely used therapeutic hormone is a well-known recombinant human erythropoietin (rHuEPO) produced from genetically engineered Chinese Hamster Ovary cells. This 34-kDa glycoprotein is produced primarily by the kidney. During the post-translational modification, glycosylation occurs with the addition of 3 N-linked (at Asn-24, -38 and -83) and one O-linked (at Ser-126) acidic oligosaccharides. rHuEPO has been used for the treatment of anemia-associated diseases including rheumatoid arthritis, AIDS, and cancer. This recombinant product is prescribed on the assumption that it will increase patients' Hb level, reduce the need for transfusion and tumor progression, and thus improve quality of life and survival. This work has established a CHO based expression system to produce active rHuEPO applicable for large scale pharmaceutical production. After cloning, selection and 4-rounds of DHFR/MTX-mediated gene amplification, the stable CHO cell lines' efficiency in secreting rHuEPO were established. The expressed rHuEPO migrated in SDS-PAGE as a broad band of 40-45 kDa which was comparable with rHuEPO reference (HEMAX®). This protein was shown to have all three N-linked glycosylation sites and its carbohydrate structure termini were capped with the functional sialic acid residuals. By analysis of in vitro interaction to its receptor (EPO-R), rHuEPO was proven to be biologically active by colorimetric MTT assay after stimulation of factor-dependent TF-1 cell proliferation in dose-dependent manner. Moreover, normocytic BALB/c mice injection was employed to confirm its bioactivity in vivo. Without any purification, the rHuEPO expressed in this study showed its principal function in physiological conditions by significantly increasing % reticulocyte in testing animal. Thus, these cell lines can be applied for large-scaled production in the biopharmaceutical industry in Thailand in the future.

Erythropoietin (EPO) เป็นฮอร์โมนที่ผลิตมาจากไต ทำงานเกี่ยวข้องกับการเร่งการ สร้างเม็ดเลือดแดงในร่างกาย recombinant human erythropoietin (rHuEPO) ที่ใช้กัน ทั่วไปซึ่งสร้างมาจาก Chinese hamster ovary (CHO) cells ด้วยเทคนิคการตัดต่อทาง พันธุกรรม เป็นฮอร์โมนบำบัดโรค EPO เป็นโปรตีนที่มีขนาด 34 kDa ซึ่งมีคาร์โบไฮเดรต เป็นส่วนประกอบสำคัญโดยในโมเลกุลประกอบด้วยโครงสร้างน้ำตาลชนิด N-linked อยู่สามตำแหน่งที่กร"