Expression, purification and characterization of a recombinant Tat47-57-Oct4 fusion protein in Pichia pastoris

Wang,Haotian; Zhang,Xinmin; Kong,Ning; Wei,Anhui; Zhang,Yanhong; Ma,Jie; Zhou,Yulai; Yan,Weiqun

doi:10.3892/mmr.2013.1857

Expression, purification and characterization of a recombinant Tat47-57-Oct4 fusion protein in Pichia pastoris

Authors:
- Haotian Wang
- Xinmin Zhang
- Ning Kong
- Anhui Wei
- Yanhong Zhang
- Jie Ma
- Yulai Zhou
- Weiqun Yan
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Affiliations: School of Pharmaceutical Sciences of Jilin University, Changchun, Jilin 130021, P.R. China, College of Basic Medical Science of China Medical University, Shenyang, Liaoning 110001, P.R. China
Published online on: December 11, 2013 https://doi.org/10.3892/mmr.2013.1857
Pages: 471-475

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Abstract

The transcription factor, Oct-4, is involved in the self-renewal of undifferentiated embryonic stem cells, and is also significant in the reprogramming process and in the development of tumors. In the present study, the fusion protein, Tat47‑57-Oct4, was secreted by the signal peptide of human serum albumin in Pichia pastoris under the control of alcohol oxidase promoter 1. The yield of recombinant Tat47‑57-Oct4 fusion protein was ~210 mg/l. Following pilot‑scale fermentation, Tat47‑57-Oct4 was purified by ammonium sulfate precipitation, Vivaflow 200 ultrafiltration and SP Sepharose fast flow chromatography in order to obtain 95.6% purity. Immunofluorescence analysis validated the ability of Tat47‑57-Oct4 to cross the cell membrane. The results demonstrated that the experimental procedure developed in the present study could produce large quantities of active Tat47‑57-Oct4 fusion protein from P. pastoris.

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