Difference between revisions of "Part:BBa K4613302"

Revision as of 13:22, 12 October 2023

pET-29a(+)-T3-M-CPA

We chose pET-29a(+) vector to express T3-ADH3 to degrade Ochratoxin A (OTA) in a more stable and efficient way.

The fusion protein consists the T3 as the protein scaffold and ADH3 as the OTA-detoxifying enzyme.

ADH3 is an amidohydrolase derived from Stenotrophomonas acidaminiphila and forms an octamer in solution.

ADH3 was reported to exhibit 57- to 35,000-fold higher activity than other enzymes and is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α (OTα) and L-β-phenylalanine (Phe).

Firstly, we achieved the solube expression of ADH3 in E. coli. We obtained the plasmid pET46_EKLIC-ADH3 from Associate Professor Longhai Dai of Hubei University. The protein of pET46_EKLIC-ADH3 was expressed by E. coli BL21(DE3) using LB medium.

After overnight incubation at 20℃, ADH3 (43.4 kDa) purified on a HiTrap Ni-NTA column. The purified protein was verified by electrophoresis on polyacrylamide gels followed by Coomassie blue staining. After that, as for ADH3, obvious target bands can be seen at 43.4 kDa shown in Fig 1b (lanes 4 and 5), confirming the successful expression of ADH3 in pET46_EKLIC vector.

Fig. 1 Results of pET46_EKLIC-ADH3. a. The plasmid map of pET46EKLIC_ADH3. b. SDS-PAGE analysis of the purified protein ADH3 in E. coli BL21(DE3) cultured in LB medium express protein for 12 hours at 20℃. Lane M: protein marker. Lanes 1-9: flow through and elution containing 10, 20, 20, 50, 50, 100, 100, 250, 250mM imidazole, respectively.

We compared the enzyme activity of CPA and ADH3. We used the methods described by Xiong L et al. (1992) to assay CPA and ADH3 activity. Fig.2 shows that the activity of CPA and ADH3. ADH3 was estimated at approximately 1.939 unit. CPA was estimated at approximately 0.646 unit. These results indicated that ADH3 exhibited 3.0-fold higher activity than CPA.

  Fig. 2 Assay of ADH3 and CPA activity. The reaction mixture containing 290 μl of 25 mM Tris buffer, 500 mM NaCl (pH 7.5), 3.26 mg/mL Hippuryl-L-phenylalanine (HLP), and 10 μl of ADH3 dissolved in 20 mM Tris-HCl (pH 8.0), 10 μl of CPA dissolved in 1 M NaCl (pH 8.4) in eppendorf tube was incubated at 25℃ for 5 min.

Moreover, we used High-Performance Liquid Chromatography (HPLC) to determine the detoxification rate of CPA and ADH3 against OTA. The HPLC chromatograms of degradation products of OTA were shown in Fig. 3. The retention times (RT) of OTA and its degradation product was 1.650 min (CPA), 1.652 min (ADH3) and 0.691 min (CPA), 0.709 min (ADH3). After the treatment of OTA with CPA and ADH3, the peak area of OTA decreased significantly compared with the control group, and the new product appeared at 0.692 min (CPA), 0.709 min (ADH3). The detoxification rates of CPA and ADH3 were 98.9% and 100%. It proved that ADH3 gave a better performance in degrading than CPA because it took less reaction time to degrade OTA completely in higher concentrations.

Fig. 3 High performance liquid chromatography (HPLC) chromatogram retention time of OTA and OTα. a.10 μg/mL OTA after incubation with methanol solution(control). b.HPLC chromatogram of degradation products of OTA after incubation with 5 U/mL M-CPA for 24 h. c. 50 μg/mL OTA after incubation with methanol solution(control). d. HPLC chromatogram of degradation products of OTA after incubation with 5 U/mL ADH3 for 30 min.

To degrade Ochratoxin A (OTA) in a more efficient way, we chose ADH3 as our final OTA-detoxifying enzyme.

We used ELPs as the backbone of the monomers. Each monomer was fused with 3 SpyTags or 3 SpyCatchers. The polymerization between these two types of monomers can proceed efficiently under multiple conditions. We linked degrading enzyme ADH3 into the SpyTag monomer to immobilize the enzyme and increase the stability of degrading enzymes.

Fig. 4 Formation of Spy Network. (a)Gene circuit. (b)The polymerization between these two types of monomers.

To verify the combination between T3 and C3, we engineered bacteria expressing T3-YFP (SpyTag-ELPs-SpyTag-ELPs-SpyTag-YFP) and bacteria expressing C3 (SpyCatcher-ELPs-SpyCatcher-ELPs-SpyCatcher). The constructed plasmids were transformed into E. Coli BL21 (DE3) and recombinant proteins were expressed using LB medium.

Purified T3-YFP and C3 were subjected to reactions under predefined time and temperature radients. The proteins after reaction were validated by electrophoresis on polyacrylamide gels (SDS-PAGE), followed by Coomassie brilliant blue staining. A distinct target band can be observed at 130 kDa, demonstrating that T3-YFP (62.4 kDa) and C3 (54.5 kDa) are capable of forming the Spy Network (Fig. 5).This reaction can occur at a variety of temperatures and has good reaction characteristics.

Fig. 5 Verification of the fabrication between T3-YFP and C3. Lane1:T3-YFP. Lane2:C3. M: Marker. Lane3: T3-YFP and C3(4℃,8h).Lane4: T3-YFP and C3(4℃,3h). Lane5: T3-YFP and C3(4℃,1h). Lane6: T3-YFP and C3(25℃,8h).Lane7: T3-YFP and C3(25℃,3h).Lane8: T3-YFP and C3(25℃,1h).Lane9: T3-YFP and C3(37℃,8h).Lane10: T3-YFP and C3(37℃,3h). Lane11: T3-YFP and C3(37℃,1h).

We constructed the fusion protein of T3-ADH3 to degrade OTA in a more efficient and stable way. We achieve the solube protein expression of T3-ADH3.

We cloned ADH3 into pET-29a(+)-T3-M-CPA vector in Fig. 6(a). T3-ADH3 were expressed by E. coli BL21(DE3) using LB medium.

After overnight incubation at 20℃, T3-ADH3 (73.6 kDa) was purified. The purified protein was verified by SDS-PAGE. After that, obvious target bands can be seen at 73.6 kDa shown in Fig 6(b) (lanes 1 and 2), respectively, confirming the successful expression of T3-ADH3. Therefore, we chose T3-ADH3 and C3 as the two monomers of the sIPN system.

Fig. 6 Results of pET46EKLIC-ADH3 and pET-29a(+)-T3-ADH3. a. The plasmid map of pET-29a(+)-T3-ADH3. b. SDS-PAGE analysis of protein expression trials in E. coli BL21(DE3) cultured in LB medium for 12 hours using pET-29a(+)-T3-ADH3. Lane M: protein marker. Lanes 1-6: flow through and elution containing 50, 50, 20, 20, 10mM imidazole, respectively.

Using T3 and C3, the formation of Semi-interpenetrating polymer network (sIPN) leads to strengthening of the mechanical property of the proteins and the versatile functionalization of the scaffold polymer by incorporating ADH3. We hope that this part and BBa_K4613012 can be associated together to make sIPN immobilized microcapsules,which can degrade OTA in wine production factory in a efficient, sustainable, and environmentally-friendly way.

Fig. 7 Immobilized microcapsules for Encapsulation of Engineered E. coli.

Reference

1. Dai Z, Yang X, Wu F, et al.Living fabrication of functional semi-interpenetrating polymeric materials[J].Nat Commun,2021, 12 (1): 3422.
2. Zakeri B, Fierer J O, Celik E, et al.Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin[J].Proc Natl Acad Sci U S A,2012, 109 (12): E690-7.
3. Reddington S C, Howarth M.Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher[J].Curr Opin Chem Biol,2015, 29: 94-9.
4. Xiong L, Peng M, Zhao M, et al.Truncated Expression of a Carboxypeptidase A from Bovine Improves Its Enzymatic Properties and Detoxification Efficiency of Ochratoxin A[J].Toxins (Basel),2020, 12 (11).

Sequence and Features