Expression Augmenting Sequence Element (EASE) Isolated From Chinese Hamster Ovary Cells

Morris, Arvia E.; Lee, Chi-Chang; Hodges, Karmen; Aldrich, Teri L.; Krantz, Carol K.; Smidt, Pauline; Thomas, Jim

doi:10.1007/978-94-011-5404-8_84

Cited by 18 publications

(20 citation statements)

References 6 publications

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“…Development of a stably transfected cell line secreting solCD39. The solCD39 cDNA insert, containing the recombinant solCD39 sequence and the IL-2 leader, was excised from the pIL-2L-solCD39 plasmid by XmaI/BglII digestion, then inserted into 2A5Ib, an expression vector optimized for stable Chinese hamster ovary (CHO) cell lines (17). The solCD39-2A5Ib plasmid was transfected into CHO cells using Lipofectamine (GIBCO BRL; Gaithersburg, MD) according to manufacturer's recommendations.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39.

Gayle

Maliszewski²,

Gimpel³

et al. 1998

J. Clin. Invest.

176

154

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Excessive platelet accumulation and recruitment, leading to vessel occlusion at sites of vascular injury, present major therapeutic challenges in cardiovascular medicine. Endothelial cell CD39, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ATP and ADP released from activated platelets, thereby abolishing recruitment. Therefore, a soluble form of CD39, retaining nucleotidase activities, would constitute a novel antithrombotic agent. We designed a recombinant, soluble form of human CD39, and isolated it from conditioned media from transiently transfected COS-1 cells and from stably transfected Chinese hamster ovary (CHO) cells. Conditioned medium from CHO cells grown under serum-free conditions was subjected to anti-CD39 immunoaffinity column chromatography, yielding a single approximately 66-kD protein with ATPase and ADPase activities. Purified soluble CD39 blocked ADP-induced platelet aggregation in vitro, and inhibited collagen-induced platelet reactivity. Kinetic analyses indicated that, while soluble CD39 had a Km for ADP of 5.9 microM and for ATP of 2.1 microM, the specificity constant kcat/Km was the same for both substrates. Intravenously administered soluble CD39 remained active in mice for an extended period of time, with an elimination phase half-life of almost 2 d. The data indicate that soluble CD39 is a potential therapeutic agent for inhibition of platelet-mediated thrombotic diatheses.

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Section: Methodsmentioning

confidence: 99%

Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39.

Gayle

Maliszewski²,

Gimpel³

et al. 1998

J. Clin. Invest.

176

154

View full text Add to dashboard Cite

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“…Our rationale for this approach was based on the following: (1) the EF-1R gene is highly expressed in most tissue types, (2) promoter, enhancer, and perhaps other elements positively regulating transcription should be located in the nearby flanking regions of the EF-1R gene, (3) Chinese hamster transcription control sequences used in vectors transfected into CHO cells would constitute a homologous expression system, and (4) CHO cells are one of the most widely used mammalian expression hosts and have an extensive history of use for expression of both research proteins and biopharmaceuticals (7). Our approach was the reverse of those in which genomic sequences associated with an integrated expression vector are cloned from a CHO cell line that exhibits high level expression of a recombinant protein (8,9). As described in this report, we found that plasmids incorporating sequences from both the 5′ and 3′ flanking regions of the CHEF1 gene are powerful expression vectors in both CHO and non-CHO mammalian cell lines.…”

Section: Introductionmentioning

confidence: 99%

High-Level Expression of Proteins in Mammalian Cells Using Transcription Regulatory Sequences from the Chinese Hamster EF-1α Gene

2004

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High-level expression of a recombinant protein in Chinese hamster ovary (CHO) cells typically requires the laborious and time-consuming procedure of stepwise gene amplification. We hypothesized that use of transcription control regions from a highly expressed gene in CHO cells to drive expression of a gene of interest might reduce the requirement for gene amplification. To this end, we cloned a 19 kb DNA fragment containing the Chinese hamster elongation factor-1alpha (EF-1alpha) gene, as well as 12 kb of 5' flanking sequence and 4 kb of 3' flanking sequence. Expression vectors containing 5' and 3' flanking sequences from the Chinese hamster EF-1alpha (CHEF1) gene were constructed and, after insertion of six different reporter genes, transfected into CHO cells. For comparison, CHO cells were also transfected with the same six reporter genes inserted into commercial vectors utilizing either the immediate early promoter from cytomegalovirus (CMV) or the human EF-1alpha promoter. The striking result from these studies was that average expression levels from pooled, stable transfectants of CHEF1 vectors were 6- to 35-fold higher than expression levels from commercial vectors that utilize the CMV or the human EF-1alpha promoters. We also used a CHEF1 vector to express a secreted and a membrane-bound protein in stably transfected non-CHO cell lines. CHEF1-driven expression of secreted alkaline phosphatase (SEAP) in three of four cell lines tested (HEK 293, K562, L1.2, and HCT 116) was 13- to 280-fold greater than that from a commercial vector employing the CMV promoter. After transfection of four different cell lines of hematopoietic origin (K562, L1.2, JY, and Jurkat), the CHEF1 vector was found to express the chemokine receptor CCR4 at >10-fold higher levels than that driven from a commercial vector utilizing the CMV promoter. Results from these experiments suggest that the CHEF1 vectors will be useful for high-level protein expression not only in CHO cells, but also in a variety of other mammalian cell lines.

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“…1). The sequence was cloned in the 2a5ib plasmid (12) for stable expression in Chinese hamster ovary cells.…”

Section: Cloning Of Monkey Genes-il-1␣mentioning

confidence: 99%

A Single Amino Acid Difference between Human and Monkey Interleukin (IL)-1β Dictates Effective Binding to Soluble Type II IL-1 Receptor

Smith

Ketchem

Moore

et al. 2002

Journal of Biological Chemistry

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Soluble type II interleukin (IL)-1 receptor (sIL1R-II)binds human IL-1␤ with high affinity and neutralizes its activity. Recombinant sIL1R-II is considered a potentially useful anti-IL-1 therapeutic, and preclinical studies have been undertaken with this molecule in primates. To better understand the cytokine-receptor interactions occurring in this nonhuman context, monkey IL-1 and IL1R-II were cloned, and their binding abilities were examined in vitro. IL-1␤ from cynomolgus monkey was capable of binding and activating the human type I IL-1 receptor. However, unlike human IL-1␤, it was unable to effectively bind and become neutralized by sIL1R-II. Human and cynomolgus IL-1␤ proteins are 96% identical, differing by only six amino acids. Structural and mutational analysis revealed that the unique sIL1R-II binding ability of human IL-1␤ is due to a single amino acid difference compared with monkey IL-1␤.

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Expression Augmenting Sequence Element (EASE) Isolated From Chinese Hamster Ovary Cells

Cited by 18 publications

References 6 publications

Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39.

Inhibition of platelet function by recombinant soluble ecto-ADPase/CD39.

High-Level Expression of Proteins in Mammalian Cells Using Transcription Regulatory Sequences from the Chinese Hamster EF-1α Gene

A Single Amino Acid Difference between Human and Monkey Interleukin (IL)-1β Dictates Effective Binding to Soluble Type II IL-1 Receptor

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