Identification of Optimal Expression Parameters and Purification of a Codon-Optimized Human GLIS1 Transcription Factor from Escherichia coli.

Abstract

GLIS1 has multiple roles in embryonic development and in deriving induced pluripotent stem cells by aiding signaling pathways and chromatin assembly. An inexpensive and simple method to produce human GLIS1 protein from Escherichia coli (E. coli) is demonstrated in this study. Various parameters such as codon usage bias, E. coli strains, media, induction conditions (such as inducer concentration, cell density, time, and temperature), and genetic constructs were investigated to obtain soluble expression of human GLIS1 protein. Using identified expression conditions and an appropriate genetic construct, the human GLIS1 protein was homogeneously purified (purity > 90%) under native conditions. Importantly, the purified protein has upheld a stable secondary structure, as demonstrated by circular dichroism spectroscopy. To the best of our knowledge, this is the first study to report the ideal expression conditions of human GLIS1 protein in E. coli to achieve soluble expression and purification under native conditions, upholding its stable secondary structure post-purification. The biological activity of the purified GLIS1 fusion protein was further assessed in MDA-MB-231 cells. This biologically active human GLIS1 protein potentiates new avenues to understand its molecular mechanisms in different cellular functions in various cancers and in the generation of induced pluripotent stem cells.

• Authors: Chandrima Dey, Vishalini Venkatesan, Rajkumar P Thummer
• Journal: Molecular biotechnology (Mol Biotechnol) Vol. 64 Issue 1 Pg. 42-56 (Jan 2022) ISSN: 1559-0305 [Electronic] United States
• PMID: 34528219 (Publication Type: Journal Article)
• Copyright: © 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.

Chemical References

• Codon
• DNA-Binding Proteins
• GLIS1 protein, human
• Recombinant Fusion Proteins
• Transcription Factors

Topics

• Cell Line, Tumor
• Cell Movement
• Cloning, Molecular
• Codon
• DNA-Binding Proteins (chemistry, genetics, isolation & purification, metabolism)
• Escherichia coli (genetics, growth & development, metabolism)
• Gene Expression
• Genetic Vectors
• Humans
• Protein Structure, Secondary
• Recombinant Fusion Proteins (chemistry, genetics, isolation & purification, metabolism)
• Solubility
• Transcription Factors (chemistry, genetics, isolation & purification, metabolism)