Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 Ni(2+)-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.
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PMID:Engineering, cloning, and functional characterization of recombinant LIM mineralization protein-1 containing an N-terminal HIV-derived membrane transduction domain. 1928 82

With the emerging role of umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) for bone regeneration and delivery of therapeutic proteins, there is an increasing need for effective gene delivery systems to modify such cells. mTAT, a TAT peptide sequence bearing histidine and cysteine residues, has been successfully used for intracellular gene delivery. Using a gWiz-GFP plasmid, we demonstrated that polyethylenimine combined with mTAT (mTAT/PEI) displayed good transfection efficacy in hUCB-MSC. hUCB-MSC transfected with mTAT/PEI were shown to express more BMP-2 protein and mRNA, indicating the feasibility of using the cells as a BMP-2 delivery system. Importantly, compared to PEI25, a "gold standard" nonviral transfection polymer, mTAT/PEI had limited toxicity to the cells. Furthermore, we demonstrated enhanced osteogenic activity in vitro for BMP-2 expressing hUCB-MSC. These results provide encouraging evidence for the potential use of mTAT/PEI to genetically modify hUCB-MSC as an approach to enhance tissue regeneration.
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PMID:Modification of Human Umbilical Cord Blood Stem Cells Using Polyethylenimine Combined with Modified TAT Peptide to Enhance BMP-2 Production. 2895 69