Part:BBa_K4003006

pET-24a-PPM1A

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.

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.

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.

Reference:

5) Wilson, R. S., Yu L Fau – Trojanowski, J. Q., Trojanowski Jq Fau – Chen, E.-Y., Chen Ey Fau – Boyle, P. A., Boyle Pa Fau – Bennett, D. A., Bennett Da Fau – Schneider, J. A. & Schneider, J. A. 2013. Tdp-43 Pathology, Cognitive Decline, And Dementia In Old Age. JAMA Neurol, 70, 1418-1424. doi: 10.1001/jamaneurol.2013.3961.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 5075
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4964
  Illegal BglII site found at 6045
  Illegal BamHI site found at 5108
  Illegal XhoI site found at 5148
  Illegal XhoI site found at 6516
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 137
  Illegal NgoMIV site found at 3184
  Illegal NgoMIV site found at 3344
  Illegal NgoMIV site found at 4932
  
• 1000
  COMPATIBLE WITH RFC[1000]