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

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.

• Authors: Haotian Wang, Xinmin Zhang, Ning Kong, Anhui Wei, Yanhong Zhang, Jie Ma, Yulai Zhou, Weiqun Yan
• Journal: Molecular medicine reports (Mol Med Rep) Vol. 9 Issue 2 Pg. 471-5 (Feb 2014) ISSN: 1791-3004 [Electronic] Greece
• PMID: 24336974 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Octamer Transcription Factor-3
• POU5F1 protein, human
• Protein Sorting Signals
• Recombinant Fusion Proteins
• Serum Albumin
• tat Gene Products, Human Immunodeficiency Virus

Topics

• Cell Differentiation (genetics)
• Cloning, Molecular
• Fermentation
• HIV-1 (genetics)
• Humans
• Octamer Transcription Factor-3 (biosynthesis, genetics)
• Pichia (genetics)
• Promoter Regions, Genetic
• Protein Sorting Signals (genetics)
• Recombinant Fusion Proteins (biosynthesis, genetics)
• Serum Albumin (genetics)
• tat Gene Products, Human Immunodeficiency Virus (biosynthesis, genetics, metabolism)