Soluble expression, purification, and secondary structure determination of human MESP1 transcription factor.

Transcription factor MESP1 is a crucial factor regulating cardiac, hematopoietic, and skeletal myogenic development. Besides, it also contributes to the generation of functional cardiomyocytes. Here, we report the soluble expression and purification of the full-length human MESP1 protein from the heterologous system, which can be delivered into the target mammalian cells. To generate this biological macromolecule, we cloned its codon-optimized gene sequence fused to a nuclear localization sequence, a cell-penetrating peptide, and a His-tag into the protein expression vector and expressed in the bacterial system (E. coli strain BL21(DE3)). Subsequently, we have screened and identified the optimal expression parameters to obtain this recombinant fusion protein in soluble form from E. coli and examined its expression concerning the placement of fusion tags at either terminal. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Notably, this purified fusion protein has maintained its secondary structure after purification, primarily comprising α-helices and random coils. This molecular tool can potentially replace its genetic and viral forms in the cardiac reprogramming of fibroblasts to induce a cardiac transcriptional profile in an integration-free manner and elucidating its role in various biological processes and diseases. KEY POINTS: • Screening of the suitable gene construct was performed and identified. • Screening of optimal expression conditions was performed and identified. • Native purification of recombinant human MESP1 protein from E. coli was performed. • Recombinant MESP1 protein has retained its secondary structure after purification.

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

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.