Difference between revisions of "Part:BBa K3190103"

Revision as of 12:11, 20 October 2019

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 localise to the endoplasmic reticulum and specifically binds to estrogen and its derivatives. The interaction between estradiol and the membrane-associated receptor triggers non-genomic signalling; intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus. For this biobrick, 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

For our studies, GPER-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.

Figure 2: Colony PCR of yeast transformed with GPER-Li-sfGFP | Specific yeast genotyping primers were used for the PCR reaction. PCR products were separated by electropheresis 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 unsuccesful chromosomal integration.

Expression of G protein-coupled estrogen receptor

Expression of the GPER-Li-sfGFP was confirmed by performing western blot, using anti GFP antibody. The results are depicted below:

Figure 3: 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-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.

Microscopy

To further verify expression of GPER-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.

Figure 4: Confocal microscopy of transformed yeast cells. A) Bright field empty vector. B) Fluorescence filter empty vector. C) Bright field GPER-sfGFP. D) Fluorescence filter GPER-sfGFP.

As expected, a clear fluorescent signal was seen in yeast expressing GPER-Li-sfGFP (Fig. 4C and D) confirming expression of GPER-Li-sfGFP. In addition, the localization of fluorescent signal (Fig. 4D) suggests localization in the endoplasmic reticulum (ER).

Sequence and Features