Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli

Abstract

Transcription factor GATA4 is expressed during early embryogenesis and is vital for proper development. In addition, it is a crucial reprogramming factor for deriving functional cardiomyocytes and was recently identified as a tumor suppressor protein in various cancers. To generate a safe and effective molecular tool that can potentially be used in a cell reprogramming process and as an anti-cancer agent, we have identified optimal expression parameters to obtain soluble expression of human GATA4 in E. coli and purified the same to homogeneity under native conditions using immobilized metal ion affinity chromatography. The identity of GATA4 protein was confirmed using western blotting and mass spectrometry. Using circular dichroism spectroscopy, it was demonstrated that the purified recombinant protein has maintained its secondary structure, primarily comprising of random coils and α-helices. Subsequently, this purified recombinant protein was applied to human cells and was found that it was non-toxic and able to enter the cells as well as translocate to the nucleus. Prospectively, this cell- and nuclear-permeant molecular tool is suitable for cell reprogramming experiments and can be a safe and effective therapeutic agent for cancer therapy.

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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. The authors gratefully acknowledge the support of DBT Program Support (Prof. S.S. Ghosh), Department of Biosciences and Bioengineering, IIT Guwahati for their assistance in Circular Dichroism experiments. This work was supported by a research grant from Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India (Early Career Research Award; ECR/2015/000193) and IIT Guwahati Institutional Start-Up Grant.

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KKH was responsible for conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing and final approval of the manuscript; PKS, SB, SHR, KR were responsible for collection and/or assembly of data, data analysis and interpretation and final editing and approval of the manuscript; 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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Fig. S1 Codon optimization analysis using Graphical codon usage analyzer. The bar graphs shown here are the codons (Codon number 51 onwards) of non-optimized and codon-optimized GATA4 gene sequences against relative adaptiveness values (details as explained in Fig. 1a legend). (JPG 4111 KB)

Fig. S2 Expression analysis, purification and characterization of the recombinant GATA4 fusion protein. a Schematic diagram of human HTN-GATA4 insert (not drawn to scale). The GATA4 gene was fused with nucleotide sequences of His tag (H) for affinity chromatography-based purification followed by TAT (T) and NLS (N) and to facilitate cell penetration and nuclear translocation in mammalian cells, respectively. b Restriction digestion analysis was performed to confirm the successful cloning of human HTN-GATA4 insert into the protein expression vector. The synthetic gene insert was cloned in the protein expression vector, pET28a(+), using restriction endonucleases, NcoI, and XhoI. The resulting plasmid, pET28a(+)-HTN-GATA4 (in short HTN-GATA4) was then confirmed by restriction digestion using various restriction enzymes, as depicted in (a) and (b). c Soluble expression analysis of HTN-GATA4 protein, induced at two different temperatures. E. coli BL21(DE3) transformed with recombinant plasmid HTN-GATA4 was induced at the early log phase (OD600 = ~0.5) with 0.25 mM of IPTG at two different temperatures (37 °C for 2 hours, and 18 °C for 24 hours). Harvested cells were lysed by ultrasonication to obtain total cell lysate (L) fraction. These fractions were then centrifuged to obtain a pellet/insoluble (P) and a soluble (S) fractions. These fractions (L, P, and S) were run on SDS-PAGE with normalized protein loading concentration of 20 μg for L fractions. Equal volume corresponding to the respective L fraction was loaded for P and S fractions*. d Purification of recombinant HTN-GATA4 protein. E. coli BL21(DE3) transformed with recombinant plasmid HTN-GATA4 was induced at the early log phase (OD600 = ~0.5) with 0.25 mM of IPTG at 37 °C for 2 hours with continuous shaking at 180 rpm. Harvested cells were lysed by ultrasonication, and the expressed protein was purified under native conditions using Ni-NTA affinity chromatography. The purification samples were run on SDS-PAGE with normalized loading volume*. e Secondary structure determination for the purified recombinant HTN-GATA4 protein. The secondary structure was determined using far UV CD spectroscopy for the purified recombinant HTN-GATA4 protein in 20 mM PB (pH 8.0 at room temperature). The CD data obtained were then analyzed, and CD spectra have been plotted with Delta Epsilon (M-1 cm-1; Y-axis) against wavelength (nM; X-axis).*The resolved polyacrylamide gel was stained with Coomassie Brilliant Blue G-250 (top) or transferred to nitrocellulose membrane and performed western blotting with His antibody (bottom). NLS: nuclear localization signal/sequence; TAT: transactivator of transcription; His tag: polyhistidine (8X); M: Protein Marker (kDa); L: Total cell lysate; P: Pellet/insoluble cell fraction; S: Soluble cell fraction; F: Flow-through fraction; W1: Wash buffer 1; W2: Wash buffer 2; W3: Wash buffer 3; E: Elution; Ab: Antibody. (TIF 5633 KB)

Fig. S3 Purification of human GATA4 fusion protein under mild denaturing conditions. E. coli BL21(DE3) transformed with recombinant plasmid GATA4-NTH was induced under optimal expression conditions (temperature: 37 °C; cell density: OD600 = ~0.5; IPTG concentration: 0.25 mM; induction time: 2 hours). Harvested cells were lysed by ultrasonication and clarified by centrifugation. Then, the clarified soluble cell fraction was treated with different concentrations of urea (0/2/4 M) at 4 °C for 12 hours with continuous shaking followed by purification using Ni-NTA affinity chromatography. a Identification of the minimal denaturant concentration required for the maximum purification yield. b Purification of GATA4-NTH under mild denaturing conditions using an optimal concentration of urea (4 M). The purification samples were run on SDS-PAGE with normalized loading volume. The resolved polyacrylamide gel was stained with Coomassie Brilliant Blue G-250 (top) or transferred to nitrocellulose membrane and performed western blotting with Histidine antibody (bottom). *Truncated GATA4-NTH protein. c Secondary structure determination for the purified recombinant GATA4-NTH protein under mild denaturing conditions. The secondary structure was determined using far UV CD spectroscopy for the purified and dialyzed recombinant GATA4-NTH protein in 20 mM PB (pH 8.0 at room temperature). The CD data obtained were then analyzed, and CD spectra have been plotted with wavelength (nM; X-axis) against Delta Epsilon (M-1 cm-1; Y-axis). M: Protein Marker (kDa); L: Total cell lysate; S: Soluble cell fraction; F: Flow-through fraction; W1: Wash buffer 1; W2: Wash buffer 2; W3: Wash buffer 3; E: Elution; Ab: Antibody (TIF 4103 KB)

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Haridhasapavalan, K.K., Sundaravadivelu, P.K., Bhattacharyya, S. et al. Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli. Bioprocess Biosyst Eng (2021). https://doi.org/10.1007/s00449-021-02516-8

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Keywords

  • GATA4
  • E. coli
  • Protein expression and purification
  • Recombinant protein
  • Secondary structure