Proof of Concept
Overview
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
![](https://static.igem.org/mediawiki/2021/6/62/T--Shanghai_Metro_Utd--Proof_of_Concept01.jpg)
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.
![](https://static.igem.org/mediawiki/2021/0/02/T--Shanghai_Metro_Utd--Proof_of_Concept02.jpg)
The pET28a-rANG was constructed.
The plates showed monoclonals of pET28a-rANG constructs.
The plates showed monoclonals of pET28a-rANG constructs.
Recombination E. coli
![](https://static.igem.org/mediawiki/2021/8/87/T--Shanghai_Metro_Utd--Proof_of_Concept03.jpg)
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.
SDS PAGE
![](https://static.igem.org/mediawiki/2021/d/dd/T--Shanghai_Metro_Utd--Proof_of_Concept04.jpg)
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
![](https://static.igem.org/mediawiki/2021/a/a3/T--Shanghai_Metro_Utd--Proof_of_Concept05.jpg)
Based on figure 5, 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, so we concentrated the solution before
and after the chromatography columns and SDS-PAGE was performed simultaneously.
![](https://static.igem.org/mediawiki/2021/3/3b/T--Shanghai_Metro_Utd--Proof_of_Concept06.jpg)
![](https://static.igem.org/mediawiki/2021/f/f0/T--Shanghai_Metro_Utd--Proof_of_Concept07.jpg)
It can be seen from Figure 6 and Figure 7 that there is actually not much
protein after the third elution.
![](https://static.igem.org/mediawiki/2021/4/4e/T--Shanghai_Metro_Utd--Proof_of_Concept08.jpg)
As seen in figure 8, the protein was successfully lyophilized into the form of
powder.
RNA Extraction
![](https://static.igem.org/mediawiki/2021/c/cf/T--Shanghai_Metro_Utd--Proof_of_Concept09.jpg)
RNA Degradation Experiment
![](https://static.igem.org/mediawiki/2021/5/5a/T--Shanghai_Metro_Utd--Proof_of_Concept10.jpg)
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).
![](https://static.igem.org/mediawiki/2021/c/cb/T--Shanghai_Metro_Utd--Proof_of_Concept11.jpg)
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.
Conclusion
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.
[1]孙德森,盛静浩.重组人血管生成素制备和生物活性鉴定[J].中国生物化学与分子生物学报,2015,31(12):1315-1321.
[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.