Part:BBa_K3190103
G protein-coupled estrogen receptor (GPER) CDS with Linker-superfolder GFP
G protein-coupled estrogen receptor (GPR30, also referred to as GPER), an intracellular transmembrane estrogen receptor, was identified in 2005 (Revankar, 2005). It is found to localize to the endoplasmic reticulum and specifically binds to estrogen and its derivatives. The interaction between estradiol and the membrane-associated receptor triggers non-genomic signaling; intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus. For this biobrick (GPER-Li-sfGFP), the C-terminal end of GPER (BBa_K3190101) was fused with superfolder GFP (BBa_K3190205) using a linker (BBa_K3190206).
Usage and Biology
In our studies, GPER-Li-sfGFP was used to examine expression and localization of GPER (BBa_K3190101) in S. cerevisiae.
Chromosomal integration
GPER-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.
Figure 1: Colony PCR of yeast transformed with GPER-Li-sfGFP | Specific yeast genotyping primers were used for the PCR reaction. PCR products were separated by electrophoresis on 1% agarose gel. The sizes of the molecular weight standards are shown on the left. Lanes 1-8 correspond to individual colonies. Expected band sizes are of 1000 bp, indicating successful chromosomal integration. Band sizes of 1500 bp indicate unsuccessful chromosomal integration.
Expression of G protein-coupled estrogen receptor
Expression of GPER-Li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:
Figure 2: Western blot of insoluble vs soluble cellular protein | Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors and GFP was used as negative and positive control respectively. Two replicate yeast cultures were used for the western blot.
As expected, GPER-Li-sfGFP was predominantly found in the insoluble fraction, suggesting possible membrane localization. The existence of a small band in the soluble fraction indicates that the protein was very abundant in the respective cells. Similarly, the presence of GFP in the insoluble fraction can be attributed to very high expression levels. However, GPER-Li-sfGFP band size of 32 kDa indicated that the receptor might have been in its truncated form.
Microscopy
To further verify expression of GPER-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.
Figure 3: Confocal microscopy of transformed yeast cells | A) Bright field empty vector. B) Fluorescence filter empty vector. C) Bright field GPER-Li-sfGFP. D) Fluorescence filter GPER-Li-sfGFP.
As expected, a clear fluorescent signal was seen in yeast expressing GPER-Li-sfGFP (Fig. 3C and D) confirming expression of GPER-Li-sfGFP. In addition, the localization of fluorescent signal (Fig. 3D) suggests localization in the endoplasmic reticulum (ER).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 750
Illegal SapI.rc site found at 1162
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