Transactivating Target Gene Expression By Recombinant SALL4B, A Pluripotent Stem Cell Marker

Objectives:Using baculovirus vector system to express recombinant TAT-SALL4B protein in large-scale. Explore the bio-activity of recombinant TAT-SALL4B in protein transduction and transactivation of target gene in vitro.Methods and Results:SALL4is an important transcription factor that supports expansion of hematopoietic stem cells. Several recent studies reveal that SALL4and SALL4B (a major isoform) are heavily modified by post-translational mechanisms and that these modifications are critical for their stability, subcellular localization, and transcriptional activities. Given the importance of SALL4and SALL4B in supporting stem cell self-renewal and expansion, we optimized a large scale expression and purification process to obtain SALL4B. Sf9insect cells were transduced with the recombinant baculovirus expressing human SALL4B fused with6xHis residues and a TAT sequence in the N-terminus. Recombinant TAT-SALL4B was purified by nickel affinity chromatography under native conditions. Immuno-blotting confirmed that recombinant SALL4B was highly expressed and purified. As the first step to test the biological activity of purified TAT-SALL4B, we investigated whether TAT-SALL4B, directly supplemented to the culture medium, was capable of entering into cells through the protein transduction process. Fluorescence microscopy showed that specific signals were present in some of the cells transduced with SALL4B protein. The signal intensity and the number of cells containing the signals were both concentration-and time-dependent, which showed that the recombinant TAT-SALL4B could enter into cells and specifically localized to the nucleus. To further test the transcriptional activity of TAT-SALL4B protein, dual luciferase reporter assay was performed. From10nM SALL4B treatment, we observed significant increases in the reporter gene expression, showing that the TAT-SALL4B protein was capable of transactivating expression of firefly luciferase driven by the OCT4promoter in a concentration-dependent manner.Conclusions:We established a large-scale expression and purification system for recombinant TAT-SALL4B.We confirmed that the purified recombinant TAT-SALL4B was biologically active. Our study thus provides an excellent platform for the potential use of SALL4B to expand hematopoietic stem cells for future clinical applications.