Team:Shanghai Metro Utd/Results

Shanghai_Metro_Utd

Results

Overview

As previously mentioned, we are interested in the property of angiogenin, and we hope to get its prokaryotic expression.

Section 1 is made up of Plasmid Extraction, Restriction Enzyme Digestion, Transformation, and Recombination. Section 2 is made up of Protein Inducible Expression, Preparation and Dissolution of Inclusion Body, Renaturation, Purification, and Activity Validation.

Results

Plasmid Construction

Figure 1: the extracted plasmid which has been used in the later part of Restriction Enzyme Digestion and Transformation.

We extracted pET-28a plasmid for the use in the later part of restriction enzyme digestion and transformation.

Figure 2: the resulting concentration of the extracted plasmid in Figure 1

We measured the concentration of the extracted pET-28a plasmid. According to the concentration, we took a certain volume for enzyme digestion.

Figure 3: Construction flowchart of the target plasmid pET-28a-rANG (by Snapgene)

Figure 4: the scanning gel electrophoresis map of the plasmid vector after enzyme digestion

As seen in figure 4, lane 1 is the pET-28a without enzyme digestion and lane 2 to 9 is the result for pET-28a after enzyme digestion. The bands of our plasmid vectors after enzyme digestion(5311bp) showed at 5000 bp around which are correct and the bands at 300 bp around were the SUMO fragments that were cut by enzyme digestion. After that, we could further conduct the E. coli transformation.

Figure 5: 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 6. E. coil having the desired pET28a-rANG

The plates in figure 6 (1) showed monoclonals of pET28a-rANG constructs.
The pET28a-rANG was constructed successfully which has been proved by sequencing as shown in figure 6 (2).

Recombination E. coli

Figure 7: 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.

SDS PAGE

Figure 8. 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 9. SDS-PAGE and stained with Coomassie Brilliant Blue after protein purifying: (1) first attempt; (2) second attempt

Based on figure 9 (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 9 (2) where you could only see one band at 15 KDa.

Figure 10. 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 11. 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 10 and Figure 11 that there is actually not much protein after the third elution.

Figure 12 Lyophilized protein

As seen in figure 12, the protein was successfully lyophilized into the form of powder.

RNA Extraction

Figure 13. The concentration of the extracted RNA by nanodrop

RNA Degradation Experiment

Figure 14. 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 15. 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.

Future Plan

As we have successfully obtained the eingineered E. coli to produce the angiogenin with well activity. We would further focus more on the improvement of the purity and yield of our protein and more tests should be conducted to ensure the safety and stability.

Reference:

[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.