A streamlined implementation of the glutamine synthetase-based protein expression system.

Journal article

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.

Authors: Knox, R; Nettleship, J; Chang, V; Hui, Z; Santos, A

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: BioMed Central
• Journal: BMC biotechnology
• Volume: 13
• Issue: 1
• Pages: 74
• Publication date: 2013-01-01
• DOI: 10.1186/1472-6750-13-74
• EISSN: 1472-6750
• ISSN: 1472-6750
• Language: English
• Keywords: Cytomegalovirus; Promoter Regions, Genetic; Cloning, Molecular; HEK293 Cells; Humans; Cricetulus; Plasmids; CHO Cells; Flow Cytometry; Transfection; Gene Expression; Animals; Green Fluorescent Proteins; Glutamate-Ammonia Ligase; Genetic Vectors; Recombinant Proteins; Cricetinae