cDNA cloning and prokaryotic expression of mouse 14–3–3 protein theta

To clone the cDNA of mouse 14-3-3θ protein gene, investigate its prokaryotic expression and produce purified recombinant mouse 14-3-3θ protein. The recombinant plasmid containing mouse 14-3-3θ gene was transformed into E. Coli BL21 to express a fusion protein with mouse 14-3-3θ protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant mouse 14-3-3θ protein. DNA sequencing confirmed that the cloned cDNA contained 738 base pairs encoding 254 amino acids, which was identical to the reported amino acid sequence of mouse 14-3-3θ. After transformation, the recombinant plasmid pGEX-14-3-3θ expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 30kDa, which was identical to the reported molecular size of mouse 14-3-3θ. Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against 14-3-3θ.