| Ren Lili,Wu Wei,Shi Yi,et al.Efficient expression and purification of Deinococcus radiodurans PprI protein in Pichia pastoris[J].Chinese Journal of Radiological Medicine and Protection,2016,36(6):406-411 |
| Efficient expression and purification of Deinococcus radiodurans PprI protein in Pichia pastoris |
| Received:November 04, 2015 |
| DOI：10.3760/cma.j.issn.0254-5098.2016.06.002 |
| KeyWords:Deinococcus radiodurans Pichia pastoris PprI protein Purification |
| FundProject:国家自然科学基金(81372922) |
| Author Name | Affiliation | E-mail | | Ren Lili | School of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China | | | Wu Wei | School of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China | | | Shi Yi | School of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China | | | Yue Ling | School of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China | | | Yang Zhanshan | School of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China | fd@suda.edu.cn |
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| Abstract:: |
| Objective To establish a technical route for the efficient expression and purification of PprI protein from Deinococcus radiodurans R1 by using eukaryotic Pichia pastoris. Methods The encoding sequence of the Deinococcus radiodurans pprI gene was modified according to the preference of Pichia pastoris' codon. Modified pprI gene was fully synthesized with PCR and a 6×His tag was added at its N-terminal. The PCR products were purified and then cloned into Pichia pastoris expression vector pHBM-905A. After utilizing Cop I and Not I double enzyme digestion and retrievering linear objective fragment, new pprI gene was transformed to the GS115 strain of Pichia pastoris. The obtained Pichia pastoris transformants were induced to express. Culture supernatants were detected by SDS-PAGE, Western blot, and mass spectrometry. A Ni-NTA column was uesd to purify the target protein and the BCA method was used to determine protein concentration. Results The coding sequence of new synthetic Deinococcus radiodurans pprI gene was correct. The purpose protein band of a molecular weight of 43000 was detected in the culture supernatant of transformed Pichia pastoris strains by SDS-PAGE and Western blot. The mass spectrometry confirmed that it was the Deinococcus radiodurans PprI protein. When the concentration of imidazole was 250 mmol/L, the elution rate of PprI protein was the highest. The purified protein concentration was 0.35 mg/ml measured by BCA method. Conclusions This study has successfully constructed a new pprI gene and the recombinant strain of Pichia pastoris secreting PprI protein, and established a technical route for the efficient expression and purification of PprI protein. |
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