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SALL4 is an important transcription factor that supports expansion of hematopoietic stem cells. Several recent studies reveal that SALL4 and 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 SALL4 and SAL4B in supporting stem cell self-renewal and expansion, we optimized a large scale expression and purification process to obtain SALL4B. Sf9 insect cells were transduced with the recombinant baculovirus expressing human SALL4B fused with 6His 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. Immuno-fluorescence microscopy revealed that recombinant TAT-SALL4B specifically localized to the nucleus in a concentration- and time-dependent manner. To further test the transcriptional activity of TAT-SALL4B protein, dual luciferase reporter assay was performed, which showed that TAT-SALL4B protein was transcriptionally active.Our results suggest that TAT-SALL4B may provide a promising cellular factor for supporting ex vivo expansion of hematopoietic stem cells. Citation Format: Mengru Yuan, Yongping Jiang, Wei Dai. Expression, purification, and characterization of recombinant TAT-SALL4B protein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 457. doi:10.1158/1538-7445.AM2014-457