The p1D4-hrGFP II expression vector: a tool for expressing and purifying visual pigments and other G protein-coupled receptors.
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 to illustrate the benefits of having a co-translated reporter during heterologous expression studies.
Morrow
James M
JM
Department of Cell & Systems Biology, University of Toronto, Room 501, Toronto, Ontario, Canada.
Chang
Belinda S W
BS
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D055358:Cone Opsins / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000096:biosynthesis / Q000235:genetics*; D013056:Spectrophotometry, Ultraviolet; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics*
D055358:Cone Opsins; D043562:Receptors, G-Protein-Coupled; D029961:Zebrafish Proteins; D012313:RNA; D012243:Rhodopsin