Difference between revisions of "Part:BBa K4613013"

Revision as of 08:10, 11 October 2023

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.

Fig.1 SDS-PAGE analysis of protein expression trials in E. coli BL21 (DE3) cultured in Terrific Broth medium for 12 hours using pQE-80L-T3-M-CPA. The temperature was 25 ℃. Lane M：protein marker. Lane 1: induced total protein. Lane 2: precipitate. Lane 3: supernatant.

Fig. 2 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°C . 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