Establishment of DHFR-deficient HEK293 cells for high yield of therapeutic glycoproteins
| Autor: | Morihisa Fujita, Xin-Yu Guo, Emmanuel Osei Mensah, Xiao-Dong Gao |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Cell Culture Techniques Bioengineering 01 natural sciences Applied Microbiology and Biotechnology law.invention 03 medical and health sciences Cricetulus law Cricetinae 010608 biotechnology Dihydrofolate reductase Protein biosynthesis Animals Humans Glycoproteins chemistry.chemical_classification biology Chinese hamster ovary cell HEK 293 cells Gene Amplification Embryonic stem cell Molecular biology Recombinant Proteins Tetrahydrofolate Dehydrogenase HEK293 Cells 030104 developmental biology chemistry Cell culture Recombinant DNA biology.protein Glycoprotein Biotechnology |
| Zdroj: | Journal of Bioscience and Bioengineering. 128:487-494 |
| ISSN: | 1389-1723 |
| DOI: | 10.1016/j.jbiosc.2019.04.005 |
| Popis: | Since the use of protein therapeutics is effective for treating intractable human diseases, the production of biologic therapeutic agents has dramatically increased over the past three decades. The Chinese hamster ovary (CHO) cell lines are the most commonly used host cell expression system for recombinant protein production. High productive and stable clonal cell lines for recombinant protein production have been established from the DHFR-deficient CHO cell using the dihydrofolate reductase/methotrexate (DHFR/MTX) selection methods. Human embryonic kidney 293 (HEK293) cells are alternative host cells widely used for protein production. In most case, however, the cells are used for the transient expression, and there is no gene amplification system in HEK293 cells. In this study, we established a DHFR-deficient HEK293 cell line for the high yield of recombinant proteins. We doubly knocked out DHFR and DHFR2 in the MAN1A1/A2/B1/C1-quadruple knockout HEK293 (QD-KO) cells, using the CRISPR/Cas9 system. The DHFR-deficient QD-KO cells were used to overexpress two proteins, lysosomal acid lipase and the constant fragment of human immunoglobulin G1 by the DHFR/MTX gene-amplification method. This method resulted in a dramatic increase in the two protein expressions in the DHFR-deficient QD-KO cells by increasing MTX concentration. Our system could be adopted in the production of several recombinant proteins including therapeutic proteins. |
| Databáze: | OpenAIRE |
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