Engineering, cloning, and functional characterization of recombinant LIM mineralization protein-1 containing an N-terminal HIV-derived membrane transduction domain

Short peptide sequences known as protein transduction domains have become increasingly prevalent as tools to internalize molecules that would otherwise remain extracellular. Here, we determine whether a purified recombinant mammalian intracellular osteogenic factor delivered by a HIV-derived TAT-peptide tag is indeed capable of intracellular localization in a form accessible to interaction with other proteins. We engineered and bacterially expressed a TAT-fusion-cDNA construct of a known osteogenic factor, LIM mineralization protein-1 (LMP-1) involved in the bone morphogenetic protein (BMP) pathway that has the potential to serve as an enhancer of BMP-2 efficacy.The expressed recombinant protein contains an N-terminal (His)₆-tag, a hemagglutinin(HA)-tag, and an 11-amino acid HIV-derived TAT-membrane transduction domain and was purified to homogeneity by Sephacryl S-100 molecular exclusion and Ni²⁺-affinity chromatography. The purified TAT–LMP-1 protein was chemically labeled with fluorescein, and its time and concentration dependent entry into rabbit blood cells was monitored by flow cytometry. We demonstrate the accumulation of TAT-tagged LMP-1 both in cytoplasmic and nuclear compartments. By performing affinity pull-down assays, we confirm our earlier findings that the recombinant TAT–LMP-1, when used as molecular bait to identify the intracellular binding proteins, interacts with Smurf1, a known binding partner of LMP-1. We also show potentiation of BMP-2 activity using the purified TAT–LMP-1 in mouse muscle C2C12 cells by assaying a heterologous luciferase-reporter construct containing multiple copies of a BMP-responsive sequence motif. Finally, we also confirm the biological activity of the purified TAT–LMP-1 by showing enhancement of BMP-2 induced increase of alkaline phosphatase mRNA and protein by RT-PCR and enzyme activity, respectively.