Team:Shanghai Metro Utd/Proof Of Concept


Proof of Concept
Angiogenin has been found to play important roles in various physiological and pathological processes, especially in neurodegenerative diseases, such as Alzheimer’s Disease (AD), Parkinson’s disease dementia (PDD), and cerebrovascular disease (CVD).
Angiogenin contributes to cell migration, invasion, vessel elongation, and neuroprotection. Produced by a spectrum of cell categories, such as the vascular endothelial cells and smooth muscle cells, and it can be recognized by the endothelial cells and elicits second messenger systems. Angiogenin is also found to be potential in curing neuro-diseases like Parkinson’s Disease and Alzheimer’s Disease.
In addition, the interaction between Angiogenin and Heparin remains to be an unsolved problem in pharmacological research.
Thus, our project aims at 1) acquiring a recombinant E. coli by introducing the plamid pET-28a-rANG that can secret functional ANG protein, which could possibly serve as an in vitro method to massive produce ANG for clinical application; 2) initially exploring the relationship between Angiogenin and Heparin, consolidating a foundation for future pharmacological research.
Supporting Experiment Results
Figure 1: The gel electrophoresis map of PCR
Lane 1 to 6 is the result of PCR. We got rANG band at around 400bp (369bp). Enzyme digestion was conducted and it was linked with digested pET-28a.
Figure 2. E. coil having the desired pET28a-rANG
The plates in figure 2(1) showed monoclonals of pET28a-rANG constructs.
The pET28a-rANG was constructed successfully which has been proved by sequencing as shown in figure 2 (2).
Recombination E. coli
Figure 3: the scanning gel electrophoresis map of Colony PCR (the top was before and the bottom was after)
Lane NC to XCN6 and lane XCN7 to N6 are the results of colony PCR. We get the band 531bp at 500bp around. It indicates that the obtained recombinant monoclonals were positive monoclonals containing the pET-28a-rANG recombinant plasmid.
Figure 4. SDS-PAGE Analyzing the proteins before and after induction, within inclusion bodies, and in supernatants.
The results for SDS-PAGE shows that the induction was successful, and the target protein (about 15KDa) was mainly in the inclusion body.
Protein Purification
Figure 5. SDS-PAGE and stained with Coomassie Brilliant Blue after protein purifying: (1) first attempt; (2) second attempt
Based on figure 5 (1), there was a band at 15 KDa before passing through the column but disappeared after passing through the column, indicating that the protein had hung the column. But we notice that there is a miscellaneous band with a small amount. In order to obtain the purified protein, we repeated the protein purification experiments and finally obtained the more purified protein as seen in figure 5 (2) where you could only see one band at 15 KDa.
Figure 6. First SDS-PAGE, Coomassie blue staining. Samples from left to right: marker; 1.before column(unconcentrated); 2. after column 1(unconcentrated); 3. after column 2(unconcentrated); 4. buffer A rinsed; 5 second elution; 6 third elution.
Figure 7. Second SDS-PAGE, Coomassie blue staining. Samples from left to right: marker; 1. second elution; 2.third elution; 3.fourth elution; 4. fifth elution.
It can be seen from Figure 6 and Figure 7 that there is actually not much protein after the third elution.
Figure 8 Lyophilized protein
As seen in figure 8, the protein was successfully lyophilized into the form of powder.
RNA Extraction
Figure 9. The concentration of the extracted RNA by nanodrop
RNA Degradation Experiment
Figure 10. electrophoresis map after RNA degradation tests
According to the electrophoresis map, the renatured rANG has ribonuclease activity with the capability of RNA incomplete degradation and produced a band at about 100 bp, which is consistent with the data in the literatures (Ref. 1, 2).
Figure 11. OD450 curve of the system with heparin and rANG
The OD450 could indicate the interaction ability between heparin and angiogenin which means that the higher the OD450 value, the greater the interaction ability between heparin and angiogenin. This curve shows the more angiogenin we added, the higher the OD450 value, namely the interaction ability between heparin and angiogenin. Therefore, it can be concluded that the interaction of heparin has dose-dependent with angiogenin.
According to the experiment results listed above, we successfully obtained a recombinant E. coli by introducing the plamid pET-28a-rANG that can secret functional ANG protein. Meanwhile, we also investigated an initial interaction between angiogenin and heparin.
[2] Shapiro R, Riordan JF, Vallee BL. Characteristic ribonucleolytic activity of human angiogenin. Biochemistry. 1986 Jun 17;25(12):3527-32. doi: 10.1021/bi00360a008. Erratum in: Biochemistry 1986 Oct 21;25(21):6730. PMID: 2424496.