Part:BBa_K3190109
Xenopus laevis lutropin-choriogonadotropic hormone receptor LHCGR CDS with Linker-superfolder GF
Mammalian Luteinizing Hormones (LH) share structural similarity, functional equivalency, and bind the same receptor as Human chorionic gonadotropin (hCG); this suggests that Xenopus laevis lutropin-choriogonadotropic hormone receptor (XLLHCGR) may serve as a good alternative to Homo sapiens LHCGR for the detection of LH, as LH has been found to induce maturation of Xenopus oocytes in vitro (Wlizla et al., 2017). For this biobrick (XLLHCGR-Li-sfGFP), the C-terminal end of XLHCGR was fused with superfolder GFP (BBa_K3190205) using a linker (BBa_K3190206).
Usage and Biology
In our studies, XLHCGR-Li-sfGFP was used to examine expression and localization of XLHCGR (BBa_K3190107) in S. cerevisiae.
Chromosomal integration
XLHCGR-Li-sfGFP was integrated into the yeast chromosome, and correct insertion was verified using colony PCR.
Figure 1: Colony PCR of yeast transformed with XLHCGR-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 XLHCGR
In order to examine expression and localization of XLHCGR, we fused sfGFP to C-terminal of the receptor using a linker and transformed yeast with the same (XLHCGR-Li-sfGFP). First, we performed western blot to verify XLHCGR expression, then we performed confocal microscopy to see intracellular localization of XLHCGR-Li-sfGFP.
Figure 2: Western blot of insoluble vs soluble cellular protein | Western blot was carried out using anti-GFP antibodies. Yeast expressing empty vectors and XLHCGR-Li-sfGFP was used as negative and positive control respectively. Two replicate yeast cultures were used for the western blot. Expected band sizes are of 107 kDa.
Since sfGFP is fused to the C-terminal end of the receptor, GFP expression confirms receptor expression. However, the band size around 32 kDa indicates that the receptor might have been in its truncated form. To further verify expression of XLHCGR-Li-sfGFP, and examine intracellular localization of the receptor, confocal microscopy was performed.
Microscopy
To further verify expression of XLHCGR-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 XLHCGR-Li-sfGFP. D) Fluorescence filter XLHCGR-Li-sfGFP.
As expected, a clear fluorescent signal was seen in yeast expressing XLHCGR-Li-sfGFP (Fig. 3C and D) confirming expression of XLHCGR-Li-sfGFP. However, localization of the fluorescent signal (Fig. 3D) was not sufficient to conclude on the specific location of XLHCGR within the cell.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 428
Illegal BglII site found at 1682 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2179
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