Part:BBa_K2354012:Experience

Applications of BBa_K2354012

Cloning of Horseradish Peroxidase

To make sure the plasmid is cloned into E. coli BL-21 strain, we extracted the plasmid from transformed E. coli.(figure 1) We validated the gene by PCR with specific primers and then we examined the result with Agarose gel electrophoresis. A successful cloning was verified from the results of a PCR performed with designed specific primers according to the theoretically expected length of horseradish peroxidase(927bp).

Figure 1.

Figure 2. Primers for VR backbone:1,3,5 ;primers for monobody:2,4,6

Expression and purification of apo-HRP and refolded-HRP

To obtain functional horseradish peroxidase we need to purify the protein from E. coli, but this kind of protein does not have any function and it is called apoprotein. After the first time purification, we refolded the protein to construct the correct structure and then we activated the apo-HRP with hemin to produce the functional HRP. We examined the existence of this protein by SDS-PAGE after purification. (figure 3)

Figure 3. SDS-PAGE for purification of HRP

Functional test of Horseradish Peroxidase

To prove the degradation ability of HRP, we mixed 25 µg of HRP with 1 mM H2O2 and BPA or NP in the 1 mL water. The environment of degradation is suitable for HRP to degrade the phenolic compounds(40℃ and pH=6-7). After degradation for 24 hours, we denatured HRP by briefly heating up to 80℃ and use LC-PDA (Liquid Chromatography - Photodiode Array detector) to analysis the result of degradation.(figure 4 and 5)

Figure 4. The result of LC-PDA (BPA sample)

Figure 5. The result of LC-PDA (NP sample)

From the result of LC-PDA, we found that there are two extra peaks showing after degradation and these extra peaks represent the by-products from degradation. Unfortunately, we can’t know how much EDCs is degraded by HRP because:

(1) The peak of the by-products overlap the peak of EDCs and we can’t get the information of remained EDCs. (There is a by-product have a broad peak from 2.2 min to 3.5min and its intensity also much higher than the peak of EDCs)

(2) The quality of the column from LC isn’t good enough to separate the by-products from EDCs due to their similar molecular properties. (This LC can’t distinguish between BPA and NP so we estimated that it can’t distinguish the degraded by-product from BPA or NP as well.)

Moreover, we use the mass spectrum to prove HRP can degrade BPA and NP by the signals of the large molecular weight of by-products.(figure 6-9) We found that there are lots of additional peaks show up after degraded by HRP, and this result can prove our HRP can degrade BPA and NP.

Figure 6. the mass spectrum of BPA

Figure 7. the mass spectrum of BPA after degradation

Figure 8. the mass spectrum of NP

Figure 9. the mass spectrum of NP after degradation

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