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


CONCLUSIONS
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.
Radcliffe Department of Medicine and MRC Human Immunology Unit, John Radcliffe Hospital, University of Oxford, Headington, OX3 9DS Oxford, UK. ray@strubi.ox.ac.uk.
Rachel
R
Knox
Joanne E
JE
Nettleship
Veronica T
VT
Chang
Zhao Kun
ZK
Hui
Ana Mafalda
AM
Santos
Nahid
N
Rahman
Ling-Pei
LP
Ho
Raymond J
RJ
Owens
Simon J
SJ
Davis
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D003587:Cytomegalovirus / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005974:Glutamate-Ammonia Ligase