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| Soluble Expression,Purification of Recombinant Aflatoxin-detoxifizyme in E.coli and Analysis of Circular Dichroism Spectrum |
| HU Rong1,4, LIU Da-ling1,3, XIE Chun-fang1,3, YAO Dong-sheng1,2 |
1. Institute of Microbial Technology, Jinan University, Guangzhou 510632, China;
2. National Engineering Research Center of Genetic Medicine, Guangzhou 510632, China;
3. GuangDong Provincial Key Laboratory of Bioengineering Medicine, Guangzhou 510632, China;
4. Guangzhou Co-win Bioengineering Co., Ltd., Guangzhou 510600, China |
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Abstract Objective: To expresss and purify the aflatoxin-detoxifizyme(ADTZ) in E.coli. and to study the biological activities and secondary structure of rADTZ. Methods and Results: the mature peptide of ADTZ was subcloned into pMAL-c2x vector to construct the prokaryotic expression plasmid. The recombinant plasmid was transformed into Rosetta(DE3) and the soluble fusion protein MBP-ADTZ was highly induced by IPTG. The expression level was approximately 50% of the total bacterial protein. The pure fusion protein was got from the cell lysate by amylose affinity chromatography. 42kDa MBP and 76kDa rADTZ were gained via digestion of fusion protein by Factor Xa, and pure rADTZ was obtained by Hydrophobic interaction chromatography(HIC). Biological activities of rADTZ had been examined and results showed that the protein had AFB1-detoxifying activity with the specific activity of 136U/mg. Circular dichroism spectra analysis revealed that rADTZ was composed of 43.3% of α-helix, 31.1% of β-sheet, 10.5% of β-turn and 15.1% of random coil. Conclusion: Purified rADTZ protein with AFB1-detoxifying activity was obtained and that laid the foundation for exploration of the relationship between the structure and function of rADTZ.
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Received: 23 November 2010
Published: 26 April 2011
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