A streamlined implementation of the glutamine synthetase-based protein expression system
Knox R, Nettleship JE, Chang VT, Hui ZK, Santos AM, Rahman N, Ho LP, Owens RJ, Davis SJ. (2013), BMC Biotechnol. 13, 74
BACKGROUND: The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultures of Chinese hamster ovary cells using this system by co-expressing green fluorescent protein from an internal ribosomal entry site and selecting for high green fluorescent protein-expressing cells using fluorescence-activated cell sorting.
RESULTS: Whereas other expression systems utilizing green fluorescent protein and fluorescence-activated cell sorting-based selection have relied on two or more sorting steps, we obtained stable expression of a test protein at levels >50% of that of an “average” clone and ~40% that of the “best” clone following a single sorting step. Versus clone-based selection, the principal savings are in the number of handling steps (reduced by a third), handling time (reduced by 70%), and the time needed to produce protein-expressing cultures (reduced by ~3 weeks). Coupling the glutamine synthetase-based expression system with product-independent selection in this way also facilitated the production of a hard-to-assay protein.
CONCLUSION: Utilizing just a single fluorescence-activated cell sorting-based selection step, the new streamlined implementation of the glutamine synthetase-based protein expression system offers protein yields sufficient for most research purposes, where <10 mg/L of protein expression is often required but relatively large numbers of constructs frequently need to be trialed.
Key figure: Comparison of conventional GS system-based, stable cell-line isolation using limiting dilution cloning, with the new protocol
Labor hours include media preparation and are based on, in the case of the conventional, cloning-based method (left), the production of 107 clonal cells, for which nine 96-well plates are plated out in addition to those needed for mock transfections. For the new protocol (right), the calculation of labor hours is based on the generation of a starting population of 107 cells obtained following the flow-cytometric sorting of three transfected 75 cm2 flasks of cells.