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Protein Transport 369 refolded correctly by cellular chaperones such as HSP90 in order to regain biological activity. 24 Following extraction from bacteria, the 6xHis-tagged recombinant proteins are purified by immobilized metal affinity chromatography, followed by ion exchange and, finally, gel filtration chromatography. Using this technique, full-length Tat fusion proteins of 15 to 121 kDa in size and spanning a wide variety of functional classes, including p16, 25 p27, 20 adenovirus E1A, HPV E7, Caspase-3, 26 HIV protease, 26 Cu,Zn-SOD, 27 eGFP, 28 Bcl-X L, 29 Rho, 30 IκB, 31 p73 dominant-negative, 32 E2F-1 dominant-negative, 33 Cre recombinase (unpublished), CRAC 34 and pRB (unpublished), have been expressed, purified and shown to be competent for cellular uptake and biological activity. The purified Tat fusion proteins can be added directly into the media or used to inject and transduce whole animals. In vitro, both primary and transformed cell types, including peripheral blood lymphocytes, diploid human fibroblasts, keratinocytes, bone marrow stem cells, osteoclasts, fibrosarcoma cells, leukemic T cells, osteosarcoma, glioma, hepatocellular carcinoma, renal carcinoma, and NIT 3T3 cells are transducible with recombinant proteins. The uptake of these proteins is rapid (
370 Cell-Penetrating Peptides: Processes and Applications An alternative method to electroporation was first described by Schutze– Redelmeier et al. 35 This group successfully transduced into T cells an antigenic peptide epitope subsequently presented at the cell surface in the MHC class I complex. Fenton et al. have also demonstrated the ability to transduce fluorescentlabeled Antp peptides into both quiescent and activated lymphocytes. 36 Our laboratory has expanded on this concept by transducing the Tat–E1A fusion protein (60 kDa) into ~100% of cells, both quiescent and cycling primary human lymphocytes in the population, in a concentration-dependent manner. The ability of Tat–E1A to bind endogenous p130 and pRB proteins (cellular targets of E1A) demonstrates that it retains its normal biological function. Moreover, this experiment was performed with a 2-year-old frozen preparation of Tat–E1A protein. Transduction has also been effective in determining the regulatory role of the small GTPase Rho in mediating integrin signaling events in avian osteoclasts. Small GTPases, such as CDC42, Rac, and Rho, regulate the cytoskeletal architecture of the cell, depending on the type of extracellular signals received. 37-39 For instance, Rho plays a dual role in formation of actin stress fibers and assembly of focal adhesions in fibroblasts. 38 Osteoclasts are important for bone remodeling processes and therefore are highly mobile cells. They achieve this state by rapidly changing their actin cytoskeletal attachment structures (called podosomes). 40,41 In vitro, these processes are stimulated by the growth factor osteopontin (OP). 42 Dissecting the role of Rho in podosome formation has been hampered by the inability to manipulate osteoclasts since these cells are essentially resistant to introduction of expression constructs by transfection or retroviral infection. However, the use of Tat-mediated transduction has allowed these barriers to be bypassed. Constitutively active and dominant-negative forms of Tat–Rho protein were generated and added to osteoclast cultures that contained the protein, based on anti-hemagglutinin (HA) immunostaining and confocal microscopy. 43 The constitutively active Tat–Rho V14 rapidly (15 to 30 min) stimulated actin stress fiber formation in a manner indistinguishable from OP during the early phases of treatment. Consistent with this finding, the dominant-negative Tat–Rho blocked the podosome-forming effects of OP. Thus, the utility of Tat-mediated transduction allows for experimentation of primary cells, such as osteoclasts, that are extremely difficult to manipulate. Another powerful example of the utility of recombinant Tat fusion protein technology was the use of Tat–Cre recombinase to modify gene expression in culture and to induce gene deletion in vivo (Becker and Dowdy, manuscript in preparation). Cre recombinase is a 38 kDa protein from bacteriophage P1 that mediates the sitespecific, intramolecular, or intermolecular recombination of DNA between a pair of 13 bp inverted repeat sequences called loxP sites; thus it permits precise deletion or activation of genes. 44-46 For example, NIH 3T3 cells containing the actin promoter separated from the β-galactosidase transgene by a loxP-’stop’-loxP motif and thereby preventing its expression were transduced with Tat–Cre protein. Unlike the poor efficiency afforded by DNA-based techniques to introduce Cre cDNA, transduction by Tat–Cre protein induced activation of β-galactosidase expression and activity in more than 95% of cells within 24 h (Figure 17.1).
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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The Tat-Derived Cell-Penetrating Pe
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24 Cell-Penetrating Peptides: Proce
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3 Transportans Margus Pooga, Mattia
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Transportans 55 Indeed, galparan is
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Transportans 57 especially the endo
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Transportans 59 In the penetration
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Transportans 61 A 21-mer antisense
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Transportans 63 Commonly, the prote
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Transportans 65 antibiotin antibodi
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Transportans 67 For cross-linking o
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Transportans 69 and still retain ef
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Model Amphipathic Peptides 73 its D
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Model Amphipathic Peptides 75 pmol/
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TABLE 4.1 Internalization of Peptid
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TABLE 4.2 Internalization of Peptid
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Model Amphipathic Peptides 81 relat
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Model Amphipathic Peptides 87 A cat
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Model Amphipathic Peptides 89 At th
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Model Amphipathic Peptides 91 16. H
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Signal Sequence-Based Cell-Penetrat
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116 Cell-Penetrating Peptides: Proc
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Interactions of Cell-Penetrating Pe
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Structure Prediction of CPPs and It
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FIGURE 9.7 (Color Figure 9.7 follow
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FIGURE 9.8 The center of the figure
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Biophysical Studies of Cell-Penetra
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Toxicity and Side Effects of Cell-P
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Kinetics of Uptake of Cell-Penetrat
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Page 350 and 351: Cell-Penetrating Peptide Conjugatio
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Page 418 and 419: Index A Abaecin, 129 Abz radiolabel
Page 420 and 421: Index 399 Diffraction, 168 Disulphi
Page 422 and 423: Index 401 structure prediction, 187
Page 424 and 425: Index 403 pRB proteins, Tat-E1A bin
Page 426 and 427: Index 405 lipid perturbation (secon
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