Difference between revisions of "Part:BBa K4003006"

Revision as of 09:37, 19 October 2021

pET-24a-PPM1A

Profile

Name: pET-24a-PPM1A

Base Pairs: 6373bp

Origin: PPM1A protein phosphatase, Mg2+/Mn2+ dependent 1A [ Homo sapiens (human)

Properties: Gene technology for protecting patented bacterial strains or cell

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.

The profiles of every basic part are as follows:

BBa_K4003002

Name: P7

Base Pairs: 19bp

Origin: T7 phage, genome

Properties: A promoter for initiation of the transcription.

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

Name: 6His

Base Pairs: 18bp

Origin: synthetic

Properties: Polyhistidine tag

Usage and Biology

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

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

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.

Sequence and Features