Citation:Liu GD,Zhou SF,Ding XC,Fang CL,Mi SY,Gao XC,Han Q.Soluble expression of recomb inant cMyc, Klf4, Oct4, and Sox2 proteins in bacteria and transduction into living cells.Int J Ophthalmol 2017;10(4):560-566,doi:10.18240/ijo.2017.04.10
Soluble expression of recomb inant cMyc, Klf4, Oct4, and Sox2 proteins in bacteria and transduction into living cells
Received:March 16, 2016  Revised:December 08, 2016
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DOI:10.18240/ijo.2017.04.10
Key Words:: reprogramming proteins; induced pluripotent stem cells; soluble expression; protein transduction
Fund Project:Supported by the Foundation of Heilongjiang Provincial Foundation for Youths Project (No.QC2011C119).
                    
AuthorInstitution
Guo-Dan Liu Department of Ophthalmology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin , Heilongjiang Province, China
Shi-Feng Zhou Department of Emergency, the First Affiliated Hospital of Harbin Medical University, Harbin , Heilongjiang Province, China
Xu-Chen Ding Department of Ophthalmology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin , Heilongjiang Province, China
Chun-Lai Fang Department of Ophthalmology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin , Heilongjiang Province, China
Shu-Yong Mi Department of Ophthalmology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin , Heilongjiang Province, China
Xiang-Chun Gao Department of Ophthalmology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin , Heilongjiang Province, China
Qing Han Department of Ophthalmology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin , Heilongjiang Province, China
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Abstract:
      AIM: To develop a new method to produce recombinant reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, in soluble format with low cost for the generation of induced pluripotent stem cells (iPSCs).

    METHODS: A short polypeptide sequence derived from the HIV trans-activator of transcription protein (TAT) and the nucleus localization signal (NLS) polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into pCold-SUMO vector which can extremely improve the solubility of recombinant proteins. Then these vector plasmids were transformed into E. coli BL21 (DE3) Chaperone competent cells for amplification. The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining. The recombinant proteins were purified by Ni-NTA resin and identified by Western blot. The transduction of these proteins into HEK 293T cells were evaluated by immunofluorescence staining.

    RESULTS: These four reprogramming proteins could be produced in soluble format in pCold-SUMO expression vector system with the assistance of chaperone proteins in bacteria. The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells.

    CONCLUSION: The results in the present study indicate the four important reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, can be produced in soluble format in bacteria with low cost. Our new method thus might be expected to greatly contribute to the future study of iPSCs.

PMC FullText Html:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406633/
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