2005
DOI: 10.1021/bi051292p
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Purification and Characterization of a Recombinant G-Protein-Coupled Receptor, Saccharomyces cerevisiae Ste2p, Transiently Expressed in HEK293 EBNA1 Cells

Abstract: The production of milligram quantities of purified, active, folded membrane protein from heterologous expression systems remains a general challenge due to intrinsically low expression levels, misfolding, and instability. Here we report the overexpression and purification of milligram quantities of functional Saccharomyces cerevisiae G-protein-coupled receptor, Ste2p, from transiently transfected human embryonic kidney 293 EBNA1 cells. Fluorescent microscopy indicates localization of Ste2p-GFP and Fc-Ste2p-GFP… Show more

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Cited by 54 publications
(54 citation statements)
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“…2A). A recent report on the purification of Ste2p heterologously expressed in HEK293 cells indicated that the purified receptor had a Kd of 34 nM for a fluorescent α-factor analog [9]. However, a direct comparison of affinity of the purified receptor between our study and that using a fluorescent ligand was not possible due to the difference in the ligands used and to the fact that the binding of the fluorescent analog was not measured in cell membranes from HEK293 cells or yeast.…”
Section: Ligand Binding Analysismentioning
confidence: 62%
See 1 more Smart Citation
“…2A). A recent report on the purification of Ste2p heterologously expressed in HEK293 cells indicated that the purified receptor had a Kd of 34 nM for a fluorescent α-factor analog [9]. However, a direct comparison of affinity of the purified receptor between our study and that using a fluorescent ligand was not possible due to the difference in the ligands used and to the fact that the binding of the fluorescent analog was not measured in cell membranes from HEK293 cells or yeast.…”
Section: Ligand Binding Analysismentioning
confidence: 62%
“…Nevertheless, the co-factors for this effect were not identified despite extensive efforts. In another study, α-factor receptor was purified using a transient expression system in HEK293 EBNA cell line [9]. About 1 mg of Ste2p was purified per liter of culture with relatively high affinity to a fluorescentlylabeled α-factor.…”
Section: Introductionmentioning
confidence: 99%
“…HC, HCF243A, HCN297Q, LC, ST6 and GT were each cloned in pTT5 vectors essentially as described previously. 60,61 The pTT vector encoding the green fluorescent protein (GFP) was used as a reporter gene and has been described elsewhere.…”
Section: Methodsmentioning
confidence: 99%
“…The Zwittergent 3-14 was purchased from Calbiochem. The codon-optimized (human codon bias) gene encoding the human ST6Gal1 protein (NP15907) intralumenal domain (aa 27-406) with a human VEGFa (P15692) signal peptide linked at its N-terminus and a C-terminal GHHHHHHHHHHG tag at its C-terminus was chemically synthesized by GenScript (Piscataway, NJ) and cloned into the pTT5 mammalian expression vector [12,13]. The secreted ST6Gal1 enzyme was expressed in CHO-EBNA1 (CHO-3E7) cells according to previously published protocols [14,15].…”
Section: Enzymes and Substratesmentioning
confidence: 99%