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.