title: Rapid amplification system for recombinant protein production in Chinese Hamster Ovary (CHO) Cells.
abstract: Recombinant therapeutic proteins have changed the face of modern medicine in the present trend and they continue to provide innovative therapies for deadly diseases. This study describes the development of a novel stable expression system for rapid amplification of genes in Chinese Hamster Ovary (CHO) cells. The expression system consists of a host CHO cell line and an expression vector (pUB-PyOri-D-C) which encodes for Polyomavirus (Py) Origin of Replication (PyOri) for amplification of integrated genes in the presence of Py Large T Antigen (PyLT) and Dihydrofolate Reductase (DHFR) selectable marker gene for selection in the presence of Methotrexate (MTX). Use of both PyOri/PyLT and DHFR can reduce the number of rounds of selection and amplification required for isolation of high producing clones. The efficiency of pUB-PyOri-D-C was compared with that of pUB-D-C plasmid using Green fluorescent protein (GFP) and Erythropoietin (EPO) as reporter proteins. Our results showed that pUB-PyOri-D-C-EPO can help development of high expressing clone in one round of selection/amplification as compared to multiple rounds of selection/amplification with pUB-D-C-EPO plasmid. CHO-DG44/EPO clone generated using pUB-PyOri-D-C-EPO gave a productivity of 119 mg/L in shake flask.
authors.affiliation: GIS, GITAM UNIVERSITY Department of Biotechnology Visakhapatnam India manojkumarmetta@gmail.com.
authors.forename: M K
authors.initials: MK
authors.lastname: Metta
authors.affiliation: GIS, GITAM UNIVERSITY Department of Biotechnology Visakhapatnam India.
authors.forename: R K
authors.initials: RK
authors.lastname: Kunaparaju
authors.affiliation: Usha Biotech Hyderabad India.
authors.forename: S
authors.initials: S
authors.lastname: Tantravahi
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin; D049452:Green Fluorescent Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate