Prokaryotic Expression and Purification of Human GST-14-3-3σ Fusion Protein and its Activity Detection

Objective: To clone prokaryotic expression vector of human 14-3-3σ gene, obtain the prokaryotic ex-pressed purified GST-14-3-3σ protein, and detect its activity. Methods: Human GST-14-3-3σ coding region wasamplified from human mammary cDNA library by PCR, and was inserted into the prokaryotic expression vectorpGEX-KG. The recombinant plasmid pGEX-KG-14-3-3σ was transformed into E.coli Rossate. The expressed prod-uct was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis. The func-tion of purified GST-14-3-3σ was identified by GST pull-down assay. Results: The DNA fragment of about 750 bp was successfully amplified by PCR, and cloned into pGEX-KG. The GST-14-3-3σ recombinant plasmid wassuccessfully obtained by double digestion identification. The inserted fragment was confirmed correct by sequenc-ing. The fusion protein of about Mr 52 000 was successfully induced, and identified by SDS-PAGE and Westernblot analysis. GST pull-down assay showed that GST-14-3-3σ could interact with AKT, which verified its knownfunction. Conclusion: The recombinant protein of GST-14-3-3σ was prokaryotic expressed and purified successful-ly, which lay the foundation for further research on cell cycle control.