Cloning,prokaryotic expression and purification of proto-oncogene Pokemon

Objective To clone mouse Pokemon gene and to induce its expression in E.coli,further to purify the recombinant protein. Methods Target gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) and cloned into pGEM-T-easy.After being identified,the recombinant plasmid was digested and cloned into prokaryotic expression vector pET-30a(+) which was digested with the same restriction enzymes.PCR,retriction enzy- me digestion and direct sequencing were used to identify the positive recombinant expression plasmid which was transformed into the E.coli BL21(DE3) and induced to express recombinant protein.The fusion protein was further purified with Ni-NTA affinity chromatography and confirmed with Western blotting. Results Restriction enzyme digestion and DNA sequencing suggested that the recombinant expression plasmid contained correct encoding region of Pokemon,without reading frame shift.SDS-PAGE demonstrated that the recombinant protein was highly expressed with the expected molecular weight at 36 000 and the purity of the fusion protein was above 90% after being purified with affinity chromatography.Western blotting also demonstrated the immune specificity of the purified fusion protein. Conclusion The prokaryotic expression vector of Pokemon is successfully constructed by gene cloning technique,and its recombinant protein with high purity was obtained.These results may play basic roles for antibody preparation and further study into the relationship between Pokemon and tumorigenesis.