title: Engineering, cloning, and functional characterization of recombinant LIM mineralization protein-1 containing an N-terminal HIV-derived membrane transduction domain.
abstract: 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.
authors.affiliation: Atlanta VA Medical Center, Department of Orthopaedics, Emory University School of Medicine, Atlanta, GA 30329, USA. ssangad@emory.edu
authors.forename: Sreedhara
authors.initials: S
authors.lastname: Sangadala
authors.forename: Motohiro
authors.initials: M
authors.lastname: Okada
authors.forename: Yunshan
authors.initials: Y
authors.lastname: Liu
authors.forename: Manjula
authors.initials: M
authors.lastname: Viggeswarapu
authors.forename: Louisa
authors.initials: L
authors.lastname: Titus
authors.forename: Scott D
authors.initials: SD
authors.lastname: Boden
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D060588:LIM Domain Proteins; D051379:Mice; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D014161:Transduction, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C484380:PDLIM7 protein, human; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus