Difference between revisions of "Part:BBa K4613302"

Revision as of 13:35, 12 October 2023

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

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

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

T3 (BBa_K4613011) and C3 (BBa_K4613012) could form protein complexes by elastin-like polypeptides (ELPs) monomers containing SpyTags and SpyCatchers. Different functional proteins can be incorporated into the polymeric scaffolds in a flexible manner due to its programmability. In this part, NAU-CHINA 2023 incorporated Mature Carboxypeptidase A (M-CPA), which is capable of hydrolyzing OTA into the non-toxic product ochratoxin α and L-α-phenylalanine (Phe) in a high degration rate. We fused M-CPA into T3 to immobilize the enzyme and increase the stability and sustainable production of M-CPA.

To verify the sIPN system, 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.4).This reaction can occur at a variety of temperatures and has good reaction characteristics.

Fig. 1 Diagram of OTA degradation principle.

We cloned T3-M-CPA (SpyTag-ELPs-SpyTag-ELPs-SpyTag-Linker-MCPA) into the PQE-80L, constructed pQE-80L-T3-M-CPA and expressed the recombinant protein in E. coli BL21(DE3) using Terrific Broth medium and 2xYT medium. After incubation at 25℃ overnight or 37℃ for 4 h and 8 h, respectively, the expression of T3-M-CPA (62.4KDa) was roughly the same as that of C3. The expression levels of both were very low. Therefore, we considered cloning T3-M-CPA into pET-29a(+) vector with the same method to try to increase the expression of T3-M-CPA.

Fig. 2 Results of PQE-80L-T3. a. The plasmid map of pQE-80l-T3. b-f. SDS-PAGE analysis of protein expression trials in E. coli BL21(DE3), their expression conditions were TB medium incubated at 37℃ for 4h, 8h, 25℃ for 12h, and 2xYT medium incubated at 37℃ for 8h, 25°C for 12 hours in turn. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitate. Lane 3: supernatant.

Fig. 3 Results of pET-29a(+)-T3-M-CPA. a. The plasmid map of pET-29a(+)-T3-M-CPA. b.SDS-PAGE analysis of the purified protein T3 in E. coli BL21(DE3) cultured in LB medium express protein for 12 hours at 20℃ . Lane M: protein marker. Lanes 1-6: flow through and elution containing 10, 50, 50, 100, 100, 250, 250 mM imidazole, respectively. c. SDS-PAGE analysis of protein expression trials in SHuffle T7 E. coli cultured in 2xYT medium for 12 hours using pQE-80L-T3. The temperature was 20℃. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitate. Lane 3: supernatant.

Fig. 4 SDS-PAGE analysis of the purified protein T3-M-CPA in E. coli BL21 (DE3) cultured in LB medium express protein for 12 hours at 20℃. Lane M: protein marker. Lanes 1-6: flow through and elution containing 10, 50, 50, 100,100,250,250 mm imidazole, respectively.

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).

  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