Part:BBa_K4003005
pro-PPM1A-His-ter
Profile
Name:Pro-PPM1A-His-ter
Base Pairs: 1312bp
Origin: PPM1A protein phosphatase, Mg2+/Mn2+ dependent 1A [ Homo sapiens (human)
Properties: Gene technology for protecting patented bacterial strains or cell
PPM1A (Protein phosphatase magnesium-dependent 1A), also known as PP2Cα, is a common protein phosphatase, and is the most characteristic and most widely studied member of the PP2C family. In the body, PPM1A can bind to a variety of proteins to dephosphorylate, and extensively regulate cell growth, immune response, ability metabolism and other life activities. The literature shows that PPM1A can reduce the expression of specific markers of type M1 inflammatory macrophages and inhibit the production of inflammatory factors. PPM1A plays a negative regulatory role in the differentiation of monocytes to macrophages.
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:
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.
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.
Microglia will produce inflammatory factors such as IL-6 and TNFα under the stimulation of LPS.
Construct design
BBa_K4003000
Name: PPM1A
Base Pairs: 1312bp
Origin: PPM1A protein phosphatase, Mg2+/Mn2+ dependent 1A [ Homo sapiens (human)
Properties: Gene technology for protecting patented bacterial strains or cell
PPM1A (Protein phosphatase magnesium-dependent 1A), also known as PP2Cα, is a common protein phosphatase, and is the most characteristic and most widely studied member of the PP2C family. In the body, PPM1A can bind to a variety of proteins to dephosphorylate, and extensively regulate cell growth, immune response, ability metabolism and other life activities. The literature shows that PPM1A can reduce the expression of specific markers of type M1 inflammatory macrophages and inhibit the production of inflammatory factors. PPM1A plays a negative regulatory role in the differentiation of monocytes to macrophages.
BBa_K4003004
Name: 6His
Base Pairs: 18bp
Origin: synthetic
Properties: Polyhistidine tag
Experimental approach
Production, purification, and SDS-PAGE analysis of recombinant PPM1A
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.
Reference:
1) Cohen, P.T.W. Overview of protein serine/threonine phosphatases. Protein Phosphatases 2004.
2) Smith SR, Schaaf K, Rajabalee N, et al. The phosphatase PPM1A controls monocyte-to-macrophage differentiation. Sci Rep. 2018; 8(1):902.
3) Hansen DV, Hanson J E, Sheng M. Microglia in alzheimer's disease. J Cell Biol 2017; 217:459-472.
4) Paolicelli, R. C., Jawaid, A., Henstridge, C. M., Valeri, A., Merlini, M., Robinson, J. L., Lee, E. B., Rose, J., Appel, S., Lee, V. M. Y., Trojanowski, J. Q., Spires-Jones, T., Schulz, P. E. & Rajendran, L. 2017. Tdp-43 Depletion In Microglia Promotes Amyloid Clearance But Also Induces Synapse Loss. Neuron, 95, 297-308.E6. doi: 10.1016/j.neuron.2017.05.037
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 752
Illegal XhoI site found at 1223 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |