Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System

Olson, Linda J.; Dahms, Nancy M.

doi:10.1007/978-1-4939-7553-2_7

Cited by 2 publications

(3 citation statements)

References 8 publications

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“…46 However, this discrepancy in post-translational modification of expressed proteins in Sf9 models has not been universally the case-especially for transmembrane proteins. 47 The molecular size of GPER transduced in Sf9 cells (Mr~55 kD; Figure 1) approximates its molecular size in mammalian systems 32 and is higher than its calculated molecular weight, which may suggest that some post-translational modification of GPER is occurring in Sf9 cells. Most importantly, the order of potency for the functional effects of GPER ligands parallels their effects reported in mammalian systems.…”

Section: Our Interpretation That [ 3 H] 2 Me Can Identify a Physiolog...mentioning

confidence: 90%

“…However, in some cases, proteins expressed using the Sf9/baculovirus system have been reported not to undergo the post‐translation modifications seen in mammalian systems which for GPER could, theoretically, affect binding characteristics 46 . However, this discrepancy in post‐translational modification of expressed proteins in Sf9 models has not been universally the case‐ especially for transmembrane proteins 47 . The molecular size of GPER transduced in Sf9 cells (Mr~55 kD; Figure 1) approximates its molecular size in mammalian systems 32 and is higher than its calculated molecular weight, which may suggest that some post‐translational modification of GPER is occurring in Sf9 cells.…”

Section: Discussionmentioning

confidence: 99%

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Correlation of functional and radioligand binding characteristics of GPER ligands confirming aldosterone as a GPER agonist

Ding¹,

Chorazyczewski

Gros

et al. 2022

Pharmacology Res & Perspec

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Aldosterone exerts some of its effects not by binding to mineralocorticoid receptors, but rather by acting via G protein‐coupled estrogen receptors (GPER). To determine if aldosterone binds directly to GPER, we studied the ability of aldosterone to compete for the binding of [3H] 2‐methoxyestradiol ([3H] 2‐ME), a high potency GPER‐selective agonist. We used GPER gene transfer to engineer Sf9‐cultured insect cells to express GPER. We chose insect cells to avoid interactions with any intrinsic mammalian receptors for aldosterone. [3H] 2‐ME binding was saturable and reversible to a high‐affinity population of receptors with Kd = 3.7 nM and Bmax = 2.2 pmol/mg. Consistent with agonist binding to G Protein‐coupled receptors, [3H] 2‐ME high‐affinity state binding was reduced in the presence of the hydrolysis‐resistant GTP analog, GppNHp. [3H] 2‐ME binding was competed for by the GPER agonist G1, the GPER antagonist G15, estradiol (E2), as well as aldosterone (Aldo). The order of potency for competing for [3H] 2‐ME binding, namely 2ME > Aldo > E2 ≥ G1, paralleled the orders of potency for inhibition of cell proliferation and inhibition of ERK phosphorylation by ligands acting at GPER. These data confirm the ability of aldosterone to interact with the GPER, consistent with the interpretation that aldosterone likely mediates its GPER‐dependent effects by direct binding to the GPER. Significance statement Despite the growing evidence for aldosterone's actions via G protein‐coupled estrogen receptors (GPER), there remains significant skepticism that aldosterone can directly interact with GPER. The current studies are the first to demonstrate directly that aldosterone indeed is capable of binding to the GPER and thus likely mediates its GPER‐dependent effects by direct binding to the receptor.

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Section: Our Interpretation That [ 3 H] 2 Me Can Identify a Physiolog...mentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Correlation of functional and radioligand binding characteristics of GPER ligands confirming aldosterone as a GPER agonist

Ding¹,

Chorazyczewski

Gros

et al. 2022

Pharmacology Res & Perspec

View full text Add to dashboard Cite

show abstract

“…The effectiveness of expressing a recombinant protein in E. coli primarily relies on the ability to prevent unfavorable interactions between newly expressed polypeptides. These interactions lead to the aggregation of intermediate folding substances instead of native protein production [ 6 , 7 , 8 , 11 ]. System efficiency can be improved by maintaining conditions that stabilize intermediate folding and promote mature structure formation.…”

Section: Introductionmentioning

confidence: 99%

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

Lee

Park

2020

Antibiotics

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Bacteria can produce recombinant proteins quickly and cost effectively. However, their physiological properties limit their use for the production of proteins in their native form, especially polypeptides that are subjected to major post-translational modifications. Proteins that rely on disulfide bridges for their stability are difficult to produce in Escherichia coli. The bacterium offers the least costly, simplest, and fastest method for protein production. However, it is difficult to produce proteins with a very large size. Saccharomyces cerevisiae and Pichia pastoris are the most commonly used yeast species for protein production. At a low expense, yeasts can offer high protein yields, generate proteins with a molecular weight greater than 50 kDa, extract signal sequences, and glycosylate proteins. Both eukaryotic and prokaryotic species maintain reducing conditions in the cytoplasm. Hence, the formation of disulfide bonds is inhibited. These bonds are formed in eukaryotic cells during the export cycle, under the oxidizing conditions of the endoplasmic reticulum. Bacteria do not have an advanced subcellular space, but in the oxidizing periplasm, they exhibit both export systems and enzymatic activities directed at the formation and quality of disulfide bonds. Here, we discuss current techniques used to target eukaryotic and prokaryotic species for the generation of correctly folded proteins with disulfide bonds.

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Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System

Cited by 2 publications

References 8 publications

Correlation of functional and radioligand binding characteristics of GPER ligands confirming aldosterone as a GPER agonist

Correlation of functional and radioligand binding characteristics of GPER ligands confirming aldosterone as a GPER agonist

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

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