Abstract:

To obtain rat His-Akt1 recombinant protein with high activity,His-Akt1-pcDNA3.1 eukaryotic expression vector was constructed firstly,and then transfected into HEK293 cells. Next,the His-Akt1 was purified by Ni column affinity chromatography（Ni-NTA）,and its activity was then identified. His-Akt1 cDNAs were amplified from the full-length recombinant Akt1-pcDNA3.1 by PCR and cloned into eukaryotic expression vector pcDNA3.1. The HEK293 cells transfected with His-Akt1-pcDNA3.1 plasmid were collected and sent to Ni-NTA. His-Akt1 purity was identified by SDS-PAGE,Coomassie blue staining and Western blot. Dialytically purified His-Akt1 finally was subjected to Western blot analysis to detect Ser473 and Thr308 phosphorylation levels. His-Akt1-pcDNA3.1 eukaryotic expression vector was constructed successfully and efficiently expressed in HEK293 cells. The Coomassie blue staining and Western blot results showed that high-purity His-Akt1 recombinant protein was obtained by purifying 2 mg overexpressing His-Akt1 protein samples with Ni-NTA and eluting with 100 mmol/L imidazole. In addition,the Ser473 and Thr308 phosphorylation（i.e. activation）of the His-Akt1 after dialysis presented both high levels. The recombinant His-Akt1-pcDNA3.1 eukaryotic expression vector is constructed successfully,and the protein highly expressed,and the purified His-Akt1 recombinant protein demonstrates high enzymatic activity.

Key words: Akt1, eukaryotic expression vector, protein purification, activity identification