The p1D4-hrGFP II expression vector: a tool for expressing and purifying visual pigments and other G protein-coupled receptors

Plasmid
James M Morrow, Belinda S W Chang

Abstract

The heterologous expression of membrane proteins such as G protein-coupled receptors can be a notoriously difficult task. We have engineered an expression vector, p1D4-hrGFP II, in order to efficiently express visual pigments in mammalian cell culture. This expression vector is based on pIRES-hrGFP II (Stratagene), with the addition of a C-terminal 1D4 epitope tag for immunoblotting and immunoaffinity purification. This vector employs the CMV promoter and hrGFP II, a co-translated reporter gene. We measured the effectiveness of pIRES-hrGFP II in expressing bovine rhodopsin, and showed a 3.9- to 5.7-fold increase in expression as measured by absorbance spectroscopy as compared with the pMT vector, a common choice for visual pigment expression. We then expressed zebrafish RH2-1 using p1D4-hrGFP II in order to assess its utility in expressing cone opsins, known to be less stable and more difficult to express than bovine rhodopsin. We show a λ(280)/λ(MAX) value of 3.3, one third of that reported in previous studies, suggesting increased expression levels and decreased levels of misfolded, non-functional visual pigment. Finally, we monitored HEK293T cell growth following transfection with pIRES-hrGFP II using fluorescence microscopy...Continue Reading

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Citations

Mar 31, 2011·Visual Neuroscience·James M MorrowBelinda S W Chang
Dec 31, 2015·Proceedings of the National Academy of Sciences of the United States of America·Ryan K SchottBelinda S W Chang
Nov 5, 2015·Evolution; International Journal of Organic Evolution·Constanze BickelmannBelinda S W Chang
Dec 17, 2014·Evolution; International Journal of Organic Evolution·Natasha I BlochTrevor D Price
Feb 19, 2016·Protein Science : a Publication of the Protein Society·Ilke van HazelBelinda S W Chang
Dec 10, 2016·Scientific Reports·Hoi Ling LukMassimo Olivucci
May 5, 2017·The Journal of Experimental Biology·Nihar BhattacharyyaBelinda S W Chang
Nov 5, 2016·The Journal of Experimental Biology·James M MorrowBelinda S W Chang
Mar 3, 2017·Proceedings. Biological Sciences·Sarah Z Dungan, Belinda S W Chang
Jan 23, 2021·Molecular Biology and Evolution·Alexander Van NynattenBelinda S W Chang
Sep 26, 2021·Molecular Biology and Evolution·Einat HauzmanBelinda S W Chang

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