Biotechnology Bulletin ›› 2020, Vol. 36 ›› Issue (12): 98-103.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0497

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Eukaryotic Expression,Purification and Activity Identification of Rat His-Akt1 Recombinant Protein

MENG Li(), DU Cai-ping()   

  1. Research Center for Biochemistry and Molecular Biology,Jiangsu Key Laboratory of Brain Disease Bioinformation,Xuzhou Medical University,Xuzhou 221004
  • Received:2020-04-28 Online:2020-12-26 Published:2020-12-22
  • Contact: DU Cai-ping E-mail:mengli@xzhmu.edu.cn;caipingdu@xzhmu.edu.cn

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