Soluble Fusion Expression, Purification Of RADTZ In E.coli And Its Analysis By Circular Dichroism Spectra

Aflatoxins were a group of strong cancerogenic mutagens with great toxicity and stability, doing much harm to human beings and animals, among which Aflatoxin B1 was the most toxic. Transformating and detoxicating of these toxins were always being concerned. Our research group has focused on biotransformating and detoxicating of aflatoxins for years, and had successfully screened out one strain of fungus named E-20 with the ablility to produce aflatoxin-detoxifizyme (ADTZ). So far, the full-length eDNA sequence had been obtained, and what's more, the recombinant protein had been expressed in Pichia. Pastoris induced by methanol.Given the relative long fermentation cycle, the need for precise control of fermentation process, there are still have difficulties to achieve high level expression of rADTZ in Pichia. Pastoris. In order to get purified enzymes simply for further researching uses, in this paper, the ORF of the rADTZ gene was cloned into the vector pMAL-C2X with a MBP tag, so the recombinant expression plasmid pMAL-C2X-ADTZ was constructed and then transformed into Rosetta(DE3), the fusion protein MBP-ADTZ was highly expressed in Rosena(DE3) induced by IPTG. The expression product displayed one significant special hybridization band in the position of 118KD in Western blot test. When inducing temperature at 37℃, MBP-ADTZ was expressed as inclusion body, while as the inducing temperature down to 20℃, MBP-ADTZ expressed was mostly soluble. After a series of optimism including adjusting IPTG concentration, IPTG adding opportunity and inducing time, expression capacity of MBP_ADTZ could be achieved up to 0.64g/L thus the theoretical expression capacity of rADTZ was 0.45g/L according to the molecular weight ratio. The pure fusion protein was got from the cell lysate by Amylose affinity chromatography. 42KD MBP and 76KD rADTZ were gained via digestion of fusion protein by Factor Xa, and pure rADTZ with a yield of 0.397g/L was got by Hydrophobic interaction chromatography(HIC) with Phenyl Sepharose 6 Fast Flow. Biological activities of rADTZ had been examined and results showed that the protein had AFB1-detoxifying activity with the detoxification rate of 19.66% and the specific activity of 26.25U/g. Circular dichroism spectra analysis revealed that rADTZ was composed of 43.3% of a-helix, 31.1% of B-sheet, 10.5% ofβ-turn and 15.1% of random coil.In the study, a prokaryotic recombinant plasmid pMAL-C2X-ADTZ was successfully constructed and effectively expressed in Rosetta(DE3) at a high level. The one- band pure rADTZ in SDS-PAGE was obtained from the cell lysate orderly via Amylose affinity chromatography, Factor Xa digestion and HIC with Phenyl Sepharose 6 Fast Flow. Experiments had proved that prokaryotic expressed rADTZ was of AFB₁-detoxifying activity and circular dichroism spectra results of rADTZ showed that the a-helix andβ-sheet contents determined was coincident with those contents predicted theoretically. This study played an important and basic role in discussing the feasibility of expressing rADTZ in prokaryotic systems.