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Journal Of Modern Oncology[ISSN:1672-4992/CN:61-1415/R]

Issue:

2016 06

Page:

865-870

Research Field:

Publishing date:

Info

Title:

The construction of prokaryotic expression vector and preparation of MDM2/4-p53 dual-target inhibitory protein

Author(s):

Dong Danfeng¹; Wang Jubo²; Wu Yinying¹; Geng Qianqian¹; Li Enxiao¹; Li Yi¹

1.Department of Oncology,The First Affiliated Hospital of Xi'an Jiaotong University,Shaanxi Xi'an 710061,China;2.Department of Neurosurgery,The Second Affiliated Hospital of Xi'an Jiaotong University£¬Shaanxi Xi'an 710004,China.

Keywords:

MDM2; MDM4; p53; thioredoxin; PTD

PACS:

R73-3

DOI:

10.3969/j.issn.1672-4992.2016.06.005

Abstract:

Objective:To prepare MDM2/4-p53 dual-target inhibitory protein with activity by molecular cloning and E.coli prokaryotic expression technology.Methods£ºThioredoxin (Trx) A was amplificated via PCR and inserted into the prokaryotic expression vector pET40b.The protein transduction domain (PTD) and the small molecule MDM2/4-p53 inhibitory peptide pDI (peptide double inhibition) were synthesized according to the reported sequence.Then the prokaryotic expression vector,which could express MDM2/4-p53 dual-target inhibitory protein,was constructed by separately inserting PTD and pDI into the C-terminal and active site of TrxA.To obtain high purity and biological MDM2/4-p53 dual-target inhibitory protein,the expression of recombinant protein was induced by IPTG in E.coli BL21 cells,the optimal expression of recombinant protein expression was analyzed,and the optimal expression condition was selected in different IPTG concentration (0.1 tendency,the tendency for 0.4L and 0.4L tendency tendency for L,1L),temperature (16¡æ,25¡æ and 37¡æ),induction time (1h,2h,3h,4h,5h,6h,7h).Then it was purified via His tag protein purification system and refolded via dialysis and redox system.Results£ºThe prokaryotic expression vector pET-TAT-TrxA-pDI expressing MDM2/4-p53 dual-target inhibitory protein was successfully constructed and transformed into E.coli BL21 cells,finally more than 60% MDM2/4-p53 dual-target inhibitory protein was obtained.The optimal condition of protein expression was 0.4mmol/L IPTG concentration and 3 hours induction time.Then the MDM2/4-p53 dual-target inhibitory protein was purified via His tag protein purification system and refolded via dialysis and redox system.The purity and the refolding rate of the recombinant protein after separately were over 99% and 80% respectively.Conclusion£ºThe MDM2/4-p53 dual-target inhibitory protein with activity was successfully prepared by molecular cloning and E.coli prokaryotic expression technology.The study provides some foundation for the further analysis of the anti-tumor activity and mechanism of MDM2/4-p53 dual-target inhibitory protein.

References:

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