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

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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.

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Acknowledgements

We thank all the members of the Laboratory for Stem Cell Engineering and Regenerative Medicine (SCERM) for their critical reading and excellent support. This work was supported by North Eastern Region—Biotechnology Program Management Cell (NERBPMC), Department of Biotechnology, Government of India (BT/PR16655/NER/95/132/2015), and also by IIT Guwahati Institutional Top-Up on Start-Up Grant.

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  1. Vishalini Venkatesan

    Present address: Universitätsmedizin Göttingen, Robert-Koch-Straße 40, 37075, Göttingen, Germany

Authors and Affiliations

  1. Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India

    Chandrima Dey, Vishalini Venkatesan & Rajkumar P. Thummer

  2. Center for Biotechnology, Anna University, Chennai, Tamil Nadu, 600025, India

    Vishalini Venkatesan

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  2. Vishalini Venkatesan
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Contributions

CD was responsible for conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript; VV was responsible for collection and/or assembly of data, data analysis and interpretation, and final editing and approval of the manuscript; and RPT was responsible for conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript, and financial support. All the authors gave consent for publication.

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Correspondence to Rajkumar P. Thummer.

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Supplementary file1 (TIF 1735 kb)

Figure S1 Schematic illustration of the tagging pattern in human GLIS1 gene. Representation of GLIS1 fusion genes tagged at both N- and C-terminal end. His polyhistidine tag (8X); TAT transactivator of transcription; NLS nuclear localization signal.

Supplementary file2 (TIF 1731 kb)

Figure S2 In silico analysis of GLIS1 gene before and after codon optimization using GRCA tool. The bar graph represents the percentage distribution of codon quality groups before (red) and after (blue) codon optimization. Codons above the threshold (green dotted line) are most likely not to interfere with the gene expression in E. coli.

Supplementary file3 (TIF 14945 kb)

Figure S3 In silico analysis of GLIS1 gene before and after codon optimization using GCUA tool. (A) Bar graph representation of the relative adaptiveness value (in %) of individual codons before (left) and after (right) codon optimization. All the 621 codons are represented for both the non-optimized and codon-optimized GLIS1 gene. Relative adaptiveness value of codon ≤ 30% is highlighted in magenta.

Supplementary file4 (TIF 4130 kb)

Figure S4 Restriction analysis of GLIS1 fusion genetic constructs. His polyhistidine tag (8X); TAT transactivator of transcription; NLS Nuclear localization signal.

Supplementary file5 (TIF 4144 kb)

Figure S5 Screening for lower inducer concentration for the expression of GLIS1-NTH fusion protein (n = 2).

Supplementary file6 (TIF 1773 kb)

Figure S6 Effect of the exogenously delivered recombinant GLIS1-NTH fusion protein on the rate of migration of BJ human fibroblast cells. Cells were seeded in 24-well culture dishes, and the respective wells were treated with protein or vehicle control for 24 h. Graphical representation of the rate of migration of protein vs. vehicle control-treated cells (p ≤ 0.05) (n = 2).

Supplementary file7 (DOCX 12 kb)

Supplementary file8 (DOCX 12 kb)

Supplementary file9 (DOCX 13 kb)

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Dey, C., Venkatesan, V. & Thummer, R.P. Identification of Optimal Expression Parameters and Purification of a Codon-Optimized Human GLIS1 Transcription Factor from Escherichia coli. Mol Biotechnol 64, 42–56 (2022). https://doi.org/10.1007/s12033-021-00390-z

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