STEP® vectors for rapid generation of stable transfected CHO cell pools and clones with high expression levels and product quality homogeneity of difficult-to-express proteins
| Autor: | Tao Jing, Sarwat Cheema, Menzo J. E. Havenga, Nathalie Veith, Femke Hoeksema, Wilfried A.M. Bakker, Yi Zhao, Remco A. Spanjaard, Lars Kober, Yiding Wang, Abhinav Luthra, Chris Yallop |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
Glycosylation Zeocin Genetic Vectors CHO Cells Transfection 01 natural sciences Iduronidase 03 medical and health sciences chemistry.chemical_compound Cricetulus Cricetinae 010608 biotechnology Animals Cloning Molecular Gene 030304 developmental biology 0303 health sciences Expression vector Chemistry Chinese hamster ovary cell Factor VII Recombinant Proteins Cell biology Internal ribosome entry site Subcloning Plasmids Biotechnology |
| Zdroj: | Protein Expression and Purification. 186:105920 |
| ISSN: | 1046-5928 |
| Popis: | Many proteins produced in CHO cells need evaluation for their clinical and commercial potential. Traditional methods based on stable clone generation are slow and unsuitable for screening larger numbers of proteins, while transient expression technologies are fast but unpredictable regarding product quality and lacking an optional path to subcloning. The STEP® vector technology introduced here combines the best properties of both methods. STEP® vectors contain a strong transcriptional cassette driving expression of a bicistronic mRNA. The gene-of-interest (GOI) is cloned upstream of a functionally impaired zeocin resistance gene (FI-Zeo) whose translation is coupled to that of the GOI through an IRES. Stable transfected cells surviving zeocin selection produce high levels of FI-Zeo and thus, high levels of the GOI-encoded protein. By using different spacers, the translational coupling efficiency and selection strength can be controlled allowing maximization of expression of any GOI. Production of laronidase and factor VII (FVII) is presented as examples of unrelated, difficult-to-express (DTE) proteins. First step is rapid generation of transfected pools with the STEP® vectors. All high expressing surviving pools showed high product quality homogeneity as did monoclonal cell lines obtained from the top pools. Up to 500 μg/mL laronidase was obtained with virtually identical glycosylation profile as reference product. For FVII, cell specific productivity of 0.45 pg/cell/day with 50 IU/μg protein matched highest reported levels of reference product even before process development. Taken together, STEP® vector technology is ideally suited for rapid, small to large-scale production of DTE proteins compared to traditional methods. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect