α2-Adrenergic receptors (α2ARs) are present in the brain and are required for memory and learning, such receptors may be a potential target for the treatment of AD.To investigate whether our target protein function is consistent with our expectations, we will explore Str KDEL_ SBP-α2A-AR-Rluc8 detached from Biotin Streptavidin Combining with Venus K-Ras, through vector construction, protein purification, cell transfection, and RUSH-BRET detection.
Experimental Procedure Flowchart
Engineering Cycles:
§ Enginering Cycle#1 Plasmid construction (DBTL)
§ Enginering Cycle#2 Protein purification (DBTL)
§ Enginering Cycle#3 RUSH-BRET (DBTL)
Explain the cycles
Ø Enginering Cycle#1 Plasmid construction
Design:
Figure1. Plasmid Map of GST-α2A-AR-CT
Figure2. Plamid Map of Str-KDEL_SBP-α2A-AR-Rluc8
Studies have shown that the 2A adrenergic receptor (2A-AR) in the G-protein-coupled receptor (GPCR) family is associated with Alzheimer's disease and is a potential target for the treatment of neurodegenerative diseases. In order to obtain the protein, firstly we will construct the GST-α2A-AR-CT plasmid and Str-KDEL_SBP-α2A-AR-Rluc8 plasmid as shown above.
Build:
1) GST-α2A-AR-CT plasmid construction
We used primers to amplify α2A-AR fragment from α2A-AR-GFP and inserted into the vectors pGEX-4T-1 to obtain GST-α2A-AR-CT plasmid.
2) Str-KDEL_SBP-α2A-AR-Rluc8 plasmid construction
First, using the human α2A-AR-Rluc8 plasmid as a template, point mutation at Sda1 to eliminate Sda1 enzyme restriction site in order to get mutant α2A-AR-Rluc8 plasmid.
Second, mutant α2A-AR-Rluc8 plasmid as a template to amplify target sequence by using primers. Gel DNA Extraction purified 2106 bp product.
Third, The plasmid Str-KDEL_SBP-EGFP-Ecadherin (purchased from Addgene) was double digested with Sda1 and Xba1 and purified the product at 5234 bp to obtain the Str-KDEL_SBP vector. we inserted the α2A-AR-Rluc8 fragment to Str-KDEL_SBP vector to obtain Str-KDEL_SBP-α2A-AR-Rluc8 plasmid.
Test:
① Colony PCR identification and cloning
We found that only three results NO.7, 10, and 11 are in line with our expectations (700 bp around). In this way, we would say these three ones are our target strips.
Figure3. Gel electrophoresis results of colony PCR: Marker-15K; 1,19,20-Negative control; 2-18-Clony PCR samples and 7,10,11 are confirmed positive
② Sending to the company for Sanger sequencing
The following alignment and base pairs which confirmed that we have obtained the expected plasmid.
Figure4. Global sequence alignment of sequencing
Learn:
Above all, we could confirm that we demonstrated the successful construction of the plasmid GST-α2A-AR-CT and Str-KDEL_SBP-α2A-AR-Rluc8, which will serve as the basis for the following experiments.
Ø Enginering Cycle#2 Protein purification
Design:
Protein purification is vital for the specification of the protein function, structure, and interactions of the protein of interest.(Protein_purification-Wikipedia, 2023) The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. In order to obtain the pure protein, we designed a GST tag attached to α2A-AR-CT so that our protein could be purified by Ni-NTA Agarose Resin.
Figure5. Schematic diagram of GST-α-AR-CT protein purification. Image by Xu Xing.
Build：
① Collect bacteria and divide the sample containing BL21(DE3) and GST-α2A-AR-CT into two 50mL centrifuges tubes
② Centrifuge three times with 4000rpm, 5 min, and 25℃
③ Fix the mold: fix two thin glass plates to the base
④ Configure the lower layer glue:2.7ml 2x lower layer buffer,55μl coagulant
⑤ Injection into the mold
⑥ Configure the upper layer glue:0.75μl upper layer gel solution,0.75μl colored upper layer gel buffer,15μl coagulant
⑦ Injection into the mold
Test:
At the end of the experiment we got the target protein by purification, and we compared it with the sample from each process during purification by using protein gel electrophoresis. The electrophoresis result showed that our target protein now became pure and we finally identified that we got the right protein (25 kDa).
Figure6. SDS-PAGE analysis of GST-α-AR-CT
Learn:
This picture is the result of SDS-PAGE which is used to test proteins. P represents precipitate; S represents cell lysate; T represents flow through; W represents wash and E represents elution. GST-α2A-AR-CT protein is found on column E, marker 25KDa, this shows GST-α2A-AR-CT protein is purified from BL21(DE3) bacteria.
The electrophoresis result showed our target protein was purified and we finally identified that we got the right protein. After that, we could further use this sample for our IP-MS and RUSH-BRET test.
Based on the IP-MS results, we could further find our candidate proteins that have the potential ability to affect the α-AR-CT transportation to the membrane.
Ø Enginering Cycle#3 RUSH-BRET
Design:
We need a way to measure the level of α2A-AR within a cell. The bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a light-emitting enzyme and a fluorescent acceptor.
Figure7. Schematic diagram of RUSH-BRET assay. Image by Xu Xing.
The Renilla luciferase Rluc8 was fused to the C-terminus of the α2AR (α2AR-Rluc8) to serve as a BRET donor. The fluorescent protein venus was fused to the N-terminus of a C-terminal fragment of KRas (V-kras) to serve as a plasma membrane-associated BRET acceptor. BRET donor and BRET acceptor interaction produced a substantial BRET signal. KDEL is connected to Streptavidin (Str), this complex was fused to the SBP-α2AR -Rluc8, KEDEL can help Str- SBP-α2AR to localize to endoplasmic reticulum (ER). Biotin can bind Str to disturb KDEL and SBP-α2AR -Rluc8 interaction, SBP-α2AR -Rluc8 will be released and transform from ER to cell membrane. On the cell membrane, Energy is transferred when Rluc8 of SBP-α2AR -Rluc8 replace the RAS of the Venus-K-RAS on the cell membrane.
Build:
In order to stimulate the transportation of α2A-AR-CT protein to the cell membrane, we used HEK293 cell lines as the host. After the cultivation, we processed cell transfection by PEI to transfer the plasmids, Str KDEL_ SBP-α2A-AR-Rluc8 and Venus-K-Ras plasmid into HEK 293 cell.
Test:
Procedure
① Add 40µL of Biotin to each well and incubate for 0, 15, 30, 1, and 3 hours. The group without biotin for 0 minutes was used as the control group
② Discard the culture medium, wash the cells twice with DPBS, add 1ml of DPBS to resuspend cells.
③ Remove a 200 μL sample of suspension to three wells in a black opaque 96-well plate and wait for 15min
④ Add 2µL Coelenterazine H to these 3 wells
⑤ Put the plate into the ELISA Microplate Reader
⑥ Record the value
⑦ BRET was processed by SubCat using (Spark® Cyto PLATE READER WITH LIVE CELL IMAGING AND REAL-TIME CYTOMETRY) and the measurement wavelength is 535nm/485nm.
BRET result
Based on the data in table 1 where the value is Venus/Rluc8, we could draw the curve as shown in Figure 8.
Table1. Fluorescence intensity of RUSH-BRET in different time
Figure8. The surface expression of α2A-AR was measured by RUSH-based BRET assays
Learn:
These increasing trends mean that the Str-KDEL_SBP-α2A-AR-Rluc8 structure has been disturbed by the biotin and α2A-AR-Rluc8 move from the endoplasmic reticulum to cell membrane. Rluc8 of SBP-α2AR -Rluc8 replaced the RAS of the Venus-K-RAS on the cell membrane and produced BRET signal that represents the amount of the protein α2A-AR on the membrane.
To conclude, our RUSH-BRET system was working to measure the amount of the protein α2A-AR on the membrane and it could be more reliable for our future research on the drug screening. Next move we will design more inquiry experiments to evaluate our RUSH-BRET system and evaluate the candidate proteins we targeted from IP-MS results. Based on our discussion with our instructors, we will test those candidate proteins by building gene knock-out cells to check if they possess the ability to affect the transportation of α2A-AR-CT to the membrane. In the future, we will try to develop more effective drugs targeted for Alzheimer's disease by targeting the alpha2A-adrenergic receptor in collaboration with pharmaceutical companies.
Protein purification (2023) Wikipedia. Available at:
https://en.wikipedia.org/wiki/Protein_purification (Accessed: 14 September 2023).
