Part:BBa_K3522001

T7 pro-His-FXR-LBD-T7 ter

This composite part is a platform to screen a small molecule FXR antagonist.

BBa_K3522001 contains T7 promoter, His tag, FXR ligand-binding domain(FXR-LBD), and T7 terminator. Farnesoid X receptor (FXR) belongs to a member of the nuclear receptor Superfamily and has been reported to be associated with glucose metabolism. For example, in FXR−/− mice, the level of hepatic gluconeogenic gene expression was decreased after fasting, while blood glucose level and the hepatic glucose production in response to pyruvate challenge were declined.

Contribution

Usage and Biology Farnesoid X receptor (FXR) is a member of metabolism related nuclear receptor family. FXR agonists have been reported to improve blood glucose levels by increasing insulin sensitivity. However, there are some side effects such as lowering the level of high density lipoprotein (HDL) and aggravating obesity after long-term administration of FXR. The antagonistic FXR can reduce body weight and blood glucose, especially can better control postprandial blood glucose. Therefore, the research of FXR antagonists is also one of the hot spots of anti-metabolic diseases. At present, the effect and mechanism of FXR on glucose metabolism are not clear. Therefore, it is important to screen a small molecule FXR antagonist as a probe to explore the mechanism of FXR regulating glucose metabolism.

Engineering Success

The BBa_K3522001 was designed to conduct FXR antagonist screening screening.

Following were our experiments and results:

Recombinant FXR ligand binding domain production and purification DNA sequence of the FXR ligand binding domain (FXR-LBD) was cloned to the pET-15b vector and transferred to the BL21 (DE3) competent cells. The transformed cells were inoculated into fresh LB medium for recombinant FXR-LBD production. When the optical density of the culture reached 0.6, IPTG at final concentration of 0.2 mM was added. The expression cells were harvested by centrifugation and broken by sonication on ice. Proteins in supernatant was purified by Ni-NTA column using an AKTA FPLC instrument according to the protocol. The protein production and purification were analyzed by SDS-PAGE. As shown in Figure 1, recombinant FXR-LBD was purified to homogeneity.

Figure 1

Figure 1. SDS-PAGE analysis of recombinant FXR-LBD production and purification. Land 1, 2, 9: protein molecular weight standards. Lane 3: the insoluble proteins in expression cells. Lane 4: the soluble proteins in expression cells. Lane 5: the proteins in the flow through of Ni-NTA column. Lane 6 and 7: eluting fractions of FXR-LBD.

Application of recombinant FXR-LBD for FXR antagonist screening FXR antagonists screened in Lab in-house compound library by AlphaScreen technology-based assay. Alphascreen technology is used for in vitro high-throughput detection of protein interactions. In the system we constructed, we used the ability of 6×His-FXR-LBD protein and Biotin-SRC1 small peptide to bind to each other, and added Nickel chelate acceptor beads and Streptavidin donor beads. When protein and small peptide are combined with each other, the distance between the two magnetic beads is close. Under the irradiation of excitation light with wavelength of 680 nm, the photosensitizer on the donor magnetic beads converts the surrounding oxygen into monomer oxygen, and the monomer oxygen diffuses to the receptor. The bulk magnetic beads activate the fluorescent groups on the acceptor magnetic beads. Furthermore, the intensity of the interaction between the protein and the small peptide was characterized by detecting the fluorescence value at the wavelength of 520-620 nm. During the operation, we dissolve 6×His-FXR-LBD protein and SRC1 small peptide on ice, and dilute to a certain concentration with assay buffer (25 mM Hepes, pH 7.4, 100 mM NaCl and 0.1% BSA). Take a white 384 ELISA plate, add 100 nM 6×His-FXR-LBD protein, 30 nM SRC1, 0.5 μM GW4064 (FXR agonist), the selected compound, and 10 μg/mL acceptor beads to each well. Afterwards, shake the plate at 300 rpm for 10 min and incubate at room temperature for 30 min. Then add the final concentration of 10 μg/mL Donor beads 300 rpm shaker for 10 minutes, and incubate for 2 hours at room temperature in the dark. Then use PerkinElmer machine to check the fluorescence value.

Figure 2A

Figure 2B

Figure 2. H7 as an FXR antagonist inhibited FXR activity.The effect of compounds (20μM) on the combination of FXR-LBD and SRC-1 was detected by AlphaScreen-based protein-peptide interaction assay. A. Among the compounds, H7 was finally selected for its highly antipathogenic activity against FXR. B. H7 antagonized GW4064-induced AlphaScreen signal. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).

As indicated in Figure 2A and 2B, compound H7 antagonized the GW4064-induced promotion of FXR-LBD binding to its coactivator SRC-1. These results thus implied the antagonistic feature of H7 against FXR. Hence, these results proved the expected functions of our BBa_K3522001.

Characterization by 2021iGEM_Nanjing_high_school

Improvement of an existing part

Compared to the old part BBa_K3522001, composite part T7 pro-His-FXR-LBD-T7 ter , we design a new part BBa_K4003006, which replaced the -FXR-LBD fragment with the PPM1A gene fragment.

Figure 8 . The blast results about the amino acid sequence of our new protein PPM1A and the old protein FXR-LBD.

PPM1A enzyme was successfully produced by transformed Escherichia coli. On the basis of purified recombinant PPM1A, an excellent PPM1A activator was obtained. The compound, compound 5, enhanced PPM1A enzyme activity significantly. Our results implied that compound 5 was a PPM1A enzymatic activator. Compound 5 has good anti-inflammatory ability on microglia. Our results are promising to alleviate the inflammatory response that occurs after microglia are activated.

Usage and Biology

Studies have shown that an Alzheimer’s patient is born every three seconds. As of 2019, more than 52 million people worldwide are living with Alzheimer’s disease. The patients in China achieved a quarter of the total. In the same year, an expert in Alzheimer’s research in the United States said,” They're maybe more people who go undiagnosed. Nowadays millions of people worldwide are suffered from the pain of Neurodegenerative diseases (NIH) and currently, no one in the world can completely cure these kinds of diseases, all we can do is slow down the process.

So our product is targeting those patients who suffer from the disease related to the microglia Inflammation, such as Alzheimer’s.

1. Purification principle of recombinant human PPM1A protein:

Fig1. ÄKTA™ pure protein purifier..

Six histidines. Histidine can specifically bind to nickel ions, and the recombinant protein with histidine tag can be adsorbed by the nickel column to achieve the purpose of purification. In addition, the eluted PPM1A protein is further separated and purified by ÄKTA™ pure protein purifier with Superdex 200 molecular sieve chromatography column. Finally, replace the storage buffer (50 mM Tris-HCl, pH 7.0, 10% glycerol, 1 mM DTT) through an ultrafiltration tube, concentrate the protein, and quickly freeze and store in liquid nitrogen.

Fig2. PPM1A enzyme activity screening principle..

pNPP (p-nitrophenyl phosphate disodium salt) is used as a substrate. When the compound and PPM1A protein are added, the enzyme activity reaction will be initiated. The absorbance at 410 nm is measured with a microplate reader. By calculating the reaction rate, the agonistic efficiency of the compound was evaluated.

Fig3. Inflammation of microglia..

Microglia will produce inflammatory factors such as IL-6 and TNFα under the stimulation of LPS.

BBa_K4003002

Usage and Biology

The promoter can react specifically to T7 RNA polymerase and is a sequence that starts gene transcription of T7 phage. It is recognized for binding and initiation of the transcription. Placed before a gene, it promotes its transcription.

BBa_K4003004

Usage and Biology

It is an polyhistidine tag, which is used in the purification of recombinant proteins

BBa_K4003000

Properties: PPM1A protein phosphatase, Mg2+/Mn2+ dependent 1A

Fig4. Plasmid diagram..

Experimental approach

Figure 5. SDS-PAGE assay..

First of all, 6×His-PPM1A was purified with a Ni-NTA column followed by AKTA FPLC according to the protocol. Then we used SDS-PAGE to test the purity of 6×His-PPM1A. As shown in Figure 1, the 6×His-PPM1A was purified successfully.

Figure 6. PPM1A enzyme activity..

Next, we screened the PPM1A activator in Lab in-house compound library by phosphatase enzyme activity assay. The effect of compounds 5 (0.01, 0.1, 1, 5, 10, 20, 40, 100 μM) on the PPM1A was detected by phosphatase activity assay with pNPP as the substrate. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).

As indicated in Figure 6, among the compounds, Compound 5 was finally selected for its highest enzymatic activity against PPM1A. Besides, it could also tell that compound 5 dose-dependently enhanced PPM1A enzyme activity. These results thus implied that compound 5 was a PPM1A enzymatic activator.

Proof of function

Figure 7. Compound 5 suppressed inflammation in BV-2 cells..

Finally, the qPCR assay was further carried out to verify the inhibitive effect of compound 5 against PPM1A. BV-2 cells were co-incubated with LPS and different concentrations (5, 10, 20 μm) of compound 5 for 24 h. Then the mRNA level of IL-1β and IL-6 were detected by the qPCR assay. All data were presented as mean±S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).

As shown in Figure 3, LPS effectively increased the mRNA level of IL-1β and IL-6, and compound 5 suppressed this increase effectively. Thus, these results confirmed that the suppressive effect of compound 5 against inflammation in BV-2 cells. Conclusion

In this project, Compound 5 was demonstrated as a PPM1A activator and its anti-inflammatory effect was determined. We will investigate the regulation of PPM1A against inflammation and the diseases related to inflammation in future scientific research work.

Future Plan

In the future, we will continue to improve the performance of drugs, further reduce the side effects of drugs and achieve less dose and greater effect at the same time. With the aggravation of global aging, our drugs will become more popular. At this time, we will strive to develop derivatives centered on neuron target drugs, which can be used to prevent and cure diseases at different stages in the early, middle, and late stages. In addition to form and efficiency, we will vigorously promote the export sales of neuron targets, expand its international influence and help more patients suffering from neurodegenerative diseases around the world.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 749
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]