title: Development of an improved mammalian overexpression method for human CD62L.
abstract: We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5-30mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4-6months to produce. To shorten the construction time, we replaced the multi-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ∼5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines.
authors.affiliation: Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, United States.
authors.forename: Haley A
authors.initials: HA
authors.lastname: Brown
authors.affiliation: Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, United States.
authors.forename: Gwynne
authors.initials: G
authors.lastname: Roth
authors.affiliation: Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, United States.
authors.forename: Genevieve
authors.initials: G
authors.lastname: Holzapfel
authors.affiliation: Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, United States.
authors.forename: Sarek
authors.initials: S
authors.lastname: Shen
authors.affiliation: Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, United States.
authors.forename: Kate
authors.initials: K
authors.lastname: Rahbari
authors.affiliation: Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, United States.
authors.forename: Joanna
authors.initials: J
authors.lastname: Ireland
authors.affiliation: Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, United States.
authors.forename: Zhongcheng
authors.initials: Z
authors.lastname: Zou
authors.affiliation: Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, United States. Electronic address: psun@nih.gov.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Sun
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D019041:L-Selectin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008717:Methionine Sulfoximine; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D019041:L-Selectin; D008717:Methionine Sulfoximine; D005974:Glutamate-Ammonia Ligase
keywords: Glutamine synthetase-based expression system; L-selectin; Methionine sulfoximine (MSX); Recombinant CD62L; Recombinant mammalian overexpression system; Stable CD62L-expression CHO cell lines