Part:BBa_K4613014

T3-ADH3

T3(BBa_K4613011) and C3(BBa_K4613012) could form protein complexes by elastin-like polypeptides(ELPs) monomers containing SpyTags and SpyCatchers. If you want to learn about the detailed introduction of T3 and C3, you can click the link below. https://parts.igem.org/Part:BBa_K4164011 https://parts.igem.org/Part:BBa_K4164012

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 ADH3, which is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α(OTα) and L-β-phenylalanine(Phe). Moreover, its soluble protein expression of ADH3 in Escherichia coli has been realized. We fused ADH3 into T3 to immobilize the enzyme and increase the stability and sustainable production of ADH3.

Fig. 2 Diagram of OTA degradation principle.

We obtained the plasmid pET46EKLIC-ADH3 from Associate Professor Longhai Dai of Hubei University, and then we cloned ADH3 into PET29a(+)-T3-M-CPA vector in Fig x(a), constructed pET2-9a(+)-T3-ADH3. The protein of pET46EKLIC-ADH3 and pET-29a(+)-T3-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 6c (lanes 4 and 5), confirming the successful expression of ADH3 in pET46EKLIC vector. For T3-ADH3, obvious target bands can be seen at 73.6 kDa, confirming the successful expression of T3-ADH3 in pET-29a(+) vector shown in Fig 6d (lanes 1 and 2).

Fig. 1 Results of pET46EKLIC-ADH3 and pET-29a(+)-T3-ADH3. a. The plasmid map of pET46EKLIC_ADH3. b. The plasmid map of pET-29a(+)-T3-ADH3. c. 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. d. 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.

To verify the sIPN system, 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.5).This reaction can occur at a variety of temperatures and has good reaction characteristics.

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. Dai L, Niu D, Huang J W, et al.Cryo-EM structure and rational engineering of a superefficient ochratoxin A-detoxifying amidohydrolase[J].J Hazard Mater,2023, 458: 131836.

Sequence and Features

Assembly Compatibility:

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

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

  Illegal NgoMIV site found at 1675
  Illegal AgeI site found at 1363
  Illegal AgeI site found at 1525
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 8