Difference between revisions of "Part:BBa K4613311"

Revision as of 08:26, 12 October 2023

pET-PC-SUMO-M-CPA

The composite part was constructed to achieve solube expression of MCPA and analyze the function of Mature Carboxypeptidase A (M-CPA). The composite part can be directly imported into plasmid and express M-CPA at the same time.

We considered adding a small ubiquitin-like modifier (SUMO) protein to further help the expression of M-CPA.

We constructed a SUMO-tag M-CPA fusion protein in pET-29a(+) vector, and expressed the protein in SHuffle T7 E. coli expression cell using 2xYT medium.

After incubation at 20℃ overnight, the soluble expression of SUMO-M-CPA (48.5 kDa) was slightly increased. However, most M-CPA still existed in precipitation. Thus, we tried to increase the expression level to obtain more soluble M-CPA.

The large expression of SUMO-M-CPA was purified and verified by SDS-PAGE. Only a very small amount of SUMO-M-CPA was found at the 48.5 kDa band shown in Fig 1b (lane 3). Therefore, we considered replacing the degrading enzyme with amidohydrolase 3 (ADH3) from Stenotrophomonas acidaminiphila.

Fig. 1 Results of pET-PC-SUMO-M-CPA. a. The plasmid map of pET-PC-SUMO-M-CPA. b. SDS-PAGE analysis of protein expression trials in SHuffle T7 E. coli cultured in 2xYT medium for 12 hours using pET-PC-SUMO-M-CPA. The temperature was 20°C. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitation. Lane 3: supernatant. c. SDS-PAGE analysis of the purified protein SUMO-M-CPA (48.5 kDa) in SHuffle T7 E. coli cultured in 2xYT medium express protein for 12 hours at 20°C. Lane M: protein marker. Lanes 1-9: flow through and elution containing 10, 10, 20, 20, 50, 50, 100, 100, 250 mM imidazole, respectively.

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.2 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. 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 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. 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.

Reference

1. 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, 2020, 12(11): 680.

Sequence and Features