Modification of a System Based on the Use of Selection and Sorting Markers for the Screening of Stable Transfectants

Abstract

The р-hook technique, which is based on protein domain expression on the cell surface, has been modified and used for the selection and highly sensitive and efficient sorting of stably transfected CHO-S cells. The model protein EYFP served as both the target protein produced by the cell and the expression marker in this study. Chimeric proteins TiBP-PDGFR and CBD-PDGFR, which enabled the binding of protein-producing cells to titanium carriers and cellulose supports, respectively, have been constructed. Combined selection with the use of the chimeric CBD-PDGFR protein exposed on the cell membrane resulted in the production of a clonal line series characterized by a high and stable level of model protein synthesis. The level of model protein expression (inferred from fluorescence) in three clones from the selected series was more than 30 times higher than the average level for the original cell pool. The expression stability in EYFP-positive clones was preserved at late (20th and 35th) culture passages. The use of the chimeric CBD-PDGFR selection and sorting marker developed in this study enables the rapid selection of highly productive stable transfectants and thus reduces the costs associated with the creation of a productive cell line collection.