Part:BBa_K4613302

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

In order to find an appropriate expression intensity to achieve balance between metabolic burden and detection efficiency, we tried the T7 lac promoter from pET-29a(+). The composite part can be directly imported into pET-29a(+) vector and express T3-M-CPA induced with IPTG.

SpyTag and SpyCatcher are a pair of reactive protein partners that can spontaneously react to reconstitute the intact folded CnaB2 domain under mild conditions. Hydrophilic elastin-like polypeptides (ELPs) composed of tandem pentapeptides of the form (VPGXG)(n) (where X may be any amino acid except proline) always serve as versatile model systems for biomaterials.

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

Fig. 1 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 (SpyCathcer-ELPs-SpyCathcer-ELPs-SpyCathcer). 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.2).This reaction can occur at a variety of temperatures and has good reaction characteristics.

Fig. 2 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 pET-29a(+)-T3-M-CPA and expressed the recombinant protein in E. coli BL21(DE3) using LB medium. After overnight incubation at 20℃, T3-M-CPA was purified on a HiTrap Ni-NTA column. The purified protein was verified by SDS-PAGE. As shown in Fig. 3b (lanes 1 and 2), T3-M-CPA mainly appear in the precipitation and almost non-existent in the supernatant, which proves that T3 formed inclusion body. We suspected that the eukaryotic origin of M-CPA leads to the formation of protein inclusion bodies. After reviewing literature, we found that the reducing conditions in the E. coli cytoplasm doesn't seem to truly favor the formation of disulfide bonds in M-CPA. To reduce the formation of inclusion body, we tried SHuffle T7 E. coli expression cell to achieve soluble expression of T3-M-CPA. The SHuffle T7 E. coli strain constitutively expresses a chromosomal copy of the disufide bond isomerase DsbC, which promotes the correction of mis-oxidized proteins into their correct form, and the cytoplasmic DsbC is also a chaperone that can assist the folding of proteins that do not require disulfide bonds. In this case, we cloned T3-M-CPA into pET-29a(+), and expressed in SHuffle T7 E. coli using 2xYT medium. After incubation at 20℃ overnight, the soluble expression of T3-M-CPA in SHuffle T7 E. coli did not increase significantly. Therefore, we considered adding a small ubiquitin-like modifier (SUMO) protein to further help the expression of T3-M-CPA.

   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-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, 20, 50, 100, 100, 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 pET-29a(+)-T3-M-CPA. The temperature was 20℃. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitation. Lane 3: supernatant.

To degrade Ochratoxin A (OTA) in a more efficient way, we chose two enzymes, Carboxypeptidase A (CPA) and ADH3. We used the methods described by Xiong L et al. (1992) to assay CPA and ADH3 activity. Fig.4 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. 4 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. 5. 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 CPA and ADH3 can degrade OTA to OTα. ADH3 gave a better performance in degrading than CPA because it took less reaction time to degrade OTA completely in higher concentrations.

Fig. 5 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.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1062
  Illegal BamHI site found at 1007
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1172
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 75