Part:BBa_K4613013

T3-M-CPA

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-M-CPA) 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 4 h, 8 h, 25℃ for 12 h, and 2xYT medium incubated at 37℃ for 8 h, 25°C for 12 h 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 h 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 h 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 h 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).

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 995
  Illegal BamHI site found at 940
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1105
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 8