TAT Protein Transduction Domain Research Articles

TFP5, a Peptide Derived from Cdk5 Activator p35, Protects Pancreatic β Cells from Glucose Toxicity.

We studied the effect of TFP5 on MIN6 cells (cultured mouse islet β cells) treated with different concentrations of glucose (5 or 25 mM). The results were verified in C57BL/6J mice (control; n=12) and db/db mice with type 2 diabetes mellitus (n=12). To synthesize TFP5, peptide p5 (a derivative of p35 protein, activator of cyclin-dependent kinase 5, Cdk5) was conjugated with a FITC tag at the N-terminus and an 11-amino acid TAT protein transduction domain at the C-terminus. TFP5 was employed to inhibit Cdk5 activity and then to evaluate its efficiency in treating experimental type 2 diabetes mellitus. TFP5 effectively inhibited the pathological hyperactivity of Cdk5, enhanced insulin secretion, and protected pancreatic β cells from apoptosis in vitro and in vivo. In addition, TFP5 inhibited inflammation in pancreatic islets by reducing the expression of inflammatory cytokines TGF-β1, TNFα, and IL-1β. These novel data indicates that TFP5 is a promising candidate for treatment of type 2 diabetes mellitus.

TAT-MeCP2 protein variants rescue disease phenotypes in human and mouse models of Rett syndrome

Rett syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants leading to functional impairment of the MeCP2 protein. Here, we used purified recombinant MeCP2e1 and MeCP2e2 protein variants fused to a TAT protein transduction domain (PTD) to evaluate their transduction ability into RTT patient-derived fibroblasts and the ability to carry out their cellular function. We then assessed their transduction ability and therapeutic effects in a RTT mouse model. In vitro, TAT-MeCP2e2-eGFP reversed the pathological hyperacetylation of histones H3K9 and H4K16, a hallmark of abolition of MeCP2 function. In vivo, intraperitoneal administration of TAT-MeCP2e1 and TAT-MeCP2e2 extended the lifespan of Mecp2−/y mice by >50%. This was accompanied by rescue of hippocampal CA2 neuron size in animals treated with TAT-MeCP2e1. Taken together, these findings provide a strong indication that recombinant TAT-MeCP2 can reach mouse brains following peripheral injection and can ameliorate the phenotype of RTT mouse models. Thus, our study serves as a first step in the development of a potentially novel RTT therapy.

Characterization of an intracellular humanized single-chain antibody to matrix protein (M1) of H5N1 virus.

We developed a human intracellular antibody based on the M1 protein from avian influenza virus H5N1 (A/meerkat/Shanghai/SH-1/2012) and then characterized the properties of this antibody. The M1 protein sequence was amplified by RT-PCR using the cDNA of the H5N1 virus as a template, expressed in bacterial expression system BL21 (DE3) and purified. A human strain, high affinity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian influenza virus. Further analysis demonstrated that TAT-HuScFv could inhibit the hemagglutination activity of the 300 TCID50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identified as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian influenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses.

TAT-MYC Recombinant Fusion Protein Enhances Hematopoietic Stem Cell Graft Performance and Immune Cell Reconstitution after Transplantation

The long interval between hematopoietic stem cell transplantation (HSCT) and hematopoietic engraftment and immune reconstitution is a dangerous time for patients. Although neutrophil counts usually rise within several weeks following stem cell transplantation, recovery of functional T and B cell repertoires can often take months; during this period patients are at risk for severe infections, and graft vs. tumor effects may be attenuated. We have examined the use of a recombinant fusion protein consisting of the N-terminal 9 amino acid segment of the HIV TAT protein transduction domain (PTD) fused to the MYC protein as a means of enhancing stem cell graft performance.Methods: Coding sequences for the 9-amino acid TAT PTD and the cDNA encoding MYC were ligated in-frame and cloned into the pET101/D-Topo vector for expression in E Coli. The fusion protein, which we designated TBX-4000, was purified to homogeneity, analyzed for contaminants, and prepared to clinical grade for administration in accordance with good manufacturing practices.Results: Following a 1-hour incubation of TBX-4000 with human cord blood cells, the chimeric protein appeared in the nucleus of target cells within 2 hours, but was rapidly metabolized, vanishing by 72 hours. Whole murine marrow and purified murine LSK+ cells that were incubated with TBX-4000 significantly out-performed control cells in competitive engraftment experiments in immune deficient recipient mice, even at ratios of 1:9 treated to control cells. Significantly higher numbers of T and B cells were recovered from both peripheral blood and splenic tissue of irradiated immune deficient mice transplanted with stem cell grafts that had been cultured for 60 minutes with TBX-4000, relative to control mice. TBX-4000 cultured cells demonstrate resistance to apoptosis induced by Granzyme B, which may account for their hardiness during engraftment in marrow niches recently exposed to the stress of pre-transplant conditioning therapy. Extensive toxicologic studies demonstrated that prolonged exposure to TBX-4000 does not cause malignant transformation of hematopoietic cells in culture, and does not lead to tumor formation in mice repeatedly injected with the recombinant protein. Mice transplanted with stem cells incubated with TBX-4000 did not develop leukemia or solid tumors during 4 months of follow-up, or following three “generations” of successive, serial transplantation. Because TBX-4000 augments immune reconstitution following hematopoietic stem cell transplantation and may stimulate graft vs. host diseases reactions, T and B cell depletion of the graft will be performed in pilot studies in humans to prevent this complication.Conclusion: Brief exposure to the TAT-MYC recombinant fusion protein enhances the performance of hematopoietic stem cells in a pre-clinical transplantation model. The brief presence of the protein protects engrafting cells from apoptotic stimuli, and does not lead to transformation of the cells following transplantation. The efficacy and safety of TBX-4000 in our pre-clinical studies serve as the basis for currently planned clinical trials of this chimeric protein in patients undergoing stem cell transplantation. DisclosuresGregory:Taiga Biotechnologies, Inc.: Employment, Equity Ownership. Turner:Taiga Biotechnologies, Inc.: Employment, Equity Ownership. Refaeli:Taiga Biotechnologies, Inc.: Employment, Equity Ownership.

Tatibody, a recombinant antibody with higher internalization potency

Using antibody drug conjugates (ADC) which can exclusively bind to their target cells and upon internalization release their toxic agent, is one of the most effective methods for killing tumor cells. Therefore, increasing the internalization rate is an important factor for tumor treatment in this case. The aim of the present study was to develop a new variant of pertuzumab (an anti-ErbB2 humanized antibody) with higher internalization rate that can be a good candidate for the production of ADC. To this end, the Human Immunodeficiency Virus TAT Protein Transduction Domain (TAT-PTD) was replaced into the structure of the pertuzumab. At first, the best site in antibody heavy chain constant region for the replacement of TAT-PTD was predicted through computational methods. Then, the resulting recombinant antibody, of which TAT-PTD was located at amino acid position 130−140 and named Tatibody, was produced in CHO-S cell line. Finally, its physicochemical properties and biological activities were evaluated and compared with pertuzumab. Results showed that the binding ability of Tatibody to the ErbB2 receptor is similar to that of pertuzumab, but its internalization potency is 3.6 fold higher and can be used as a good candidate for ADC construction.

The TAT Protein Transduction Domain as an Intra-Articular Drug Delivery Technology.

Intra-articular drug delivery holds great promise for the treatment of joint diseases such as osteoarthritis. The objective of this study was to evaluate the TAT peptide transduction domain (TAT-PTD) as a potential intra-articular drug delivery technology for synovial joints. Experiments examined the ability of TAT conjugates to associate with primary chondrocytes and alter cellular function both in vitro and in vivo. Further experiments examined the ability of the TAT-PTD to bind to human osteoarthritic cartilage. The results show that the TAT-PTD associates with chondrocytes, is capable of delivering siRNA for chondrocyte gene knockdown, and that the recombinant enzyme TAT-Cre is capable of inducing in vivo genetic recombination within the knee joint in a reporter mouse model. Last, binding studies show that osteoarthritic cartilage preferentially uptakes the TAT-PTD from solution. The results suggest that the TAT-PTD is a promising delivery strategy for intra-articular therapeutics.

Newly synthesized peptide, Ara-27, exhibits significant improvement in cell-penetrating ability compared to conventional peptides.

Cell-penetrating peptides (CPPs) are short amino acid sequences known to act as a vehicle for enhancing the intracellular translocating efficiency of extracellular molecules. Although many groups have attempted to develop peptides with high cell-penetrating efficiencies, very few have demonstrated efficient cellular uptake of CPPs at low concentrations. Here, we describe a newly synthesized peptide derived from Arabidopsis, Ara-27, which exhibits significant improvement in cell-penetrating efficiency compared to existing CPPs. The cell-penetrating efficiency of Ara-27 was compared with the commonly used Tat-protein transduction domain (Tat-PTD) and membrane translocating sequence (MTS) in human dermal fibroblast (HDF) and human dental pulp stem cells (hDPSC). Cell-penetrating efficiency of fluorescein isothiocyanate (FITC)-labeled CPPs were assessed by flow cytometry and visualized by confocal microscopy. Flow cytometric analysis revealed >99% cell-penetrating efficiency for 2 μM Ara-27 in both HDF and hDPSC. In contrast, 2 μM Tat-PTD and MTS showed <10% cell-penetrating efficiency in both cells. In support, relative fluorescence intensities of FITC-labeled Ara-27 were around 8 to 22 times higher than those of Tat-PTD and MTS in both cells. Confocal analysis revealed internalization of 0.2 and 2 μM Ara-27 in both human cells, which was not observed for Tat-PTD and MTS at either concentration. In conclusion, this study describes a novel CPP, Ara-27, which exhibit significant improvement in intracellular uptake compared to conventional CPPs, without affecting cell viability. Thus, development of Ara-27 based peptides may lead to improved delivery of functional cargo such as small molecules, siRNA, and drugs for in vivo studies.

Mn-TAT PTD-Ngb attenuates oxidative injury by an enhanced ROS scavenging ability and the regulation of redox signaling pathway

Neurological diseases have a close relationship to excessive reactive oxygen species (ROS). Neuroglobin (Ngb), an intrinsic protective factor, protected cells from hypoxic/ischemic injury. In the present, we reported a novel neuroprotective manganese porphyrin reconstituted metal protein, Mn-TAT PTD-Ngb, consisting of a HIV Tat protein transduction domain sequence (TAT PTD) attached to the N-terminal of apo-Ngb. Mn-TAT PTD-Ngb had a stronger ROS scavenging ability than that of TAT PTD-Ngb, and reduced intracellular ROS production and restored the function of the mitochondria and inhibited the mitochondria-dependent apoptosis. Besides, Mn-TAT PTD-Ngb activated the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway, which up-regulated the expression of nuclear factor E2-related factor 2 (Nrf2), Heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase (CAT). The results showed that the redox chemistry of Mn-TAT PTD-Ngb and redox regulation of multiple signaling pathways attenuated the oxidative injury.

Development of Safe and Non-Self-Immunogenic Mucosal Adjuvant by Recombinant Fusion of Cholera Toxin A1 Subunit with Protein Transduction Domain.

Potential use of cholera toxin (CT) as a mucosal vaccine adjuvant has been documented in a variety of animal models. However, native CT is highly toxic to be used as a mucosal adjuvant in humans. Here, we demonstrate a new approach to generate a mucosal adjuvant by replacing the B subunit of CT with HIV-1 Tat protein transduction domain (PTD), which efficiently delivers fusion proteins into the cell cytoplasm by unspecific binding to cell surface. We compared the adjuvanticity and toxicity of Tat PTD-CTA1-Tat PTD (TCTA1T) with those of CT. Our results indicate that intranasal (i.n.) delivery of ovalbumin (OVA) with TCTA1T significantly augments the OVA-specific systemic and mucosal antibody responses to levels comparable to those seen with CT adjuvant. Moreover, in vivo cytotoxic T lymphocyte activity elicited by TCTA1T was significantly higher than that elicited by a mutant TCTA1T (TmCTA1T) lacking ADP-ribosyltransferase function. In addition, coadministration of influenza M2 protein with TCTA1T conferred near complete protection against lethal influenza virus challenge. Importantly, TCTA1T, in contrast to CT, did not induce serum IgG antibody responses to itself and was shown to be nontoxic. These results suggest that TCTA1T may be a safe and effective adjuvant when given by mucosal routes.

Porcine Mx1 fused to HIV Tat protein transduction domain (PTD) inhibits classical swine fever virus infection in vitro and in vivo.

BackgroundClassical swine fever (CSF) caused by CSF virus (CSFV) is highly contagious andcauses significant economic losses in the pig industry throughout the world. Previously we demonstrated that porcine Mx1 (poMx1), when fused to HIV Tat protein transduction domain (PTD), inhibits CSFV propagation in PK-15 cells, but it is unknown whether PTD-poMx1 exhibits antiviral activity in other porcine lines and it is efficacious for controlling CSFV infection in pigs in China.MethodsTwo porcine cell lines, ST and 3D4/21, were used to investigate in vitro antiviral activity of PTD-poMx1 against CSFV using confocal microscopy, western blot, flow cytometry, and real-time RT-PCR. Furthermore, in vivo antiviral activity of PTD-poMx1 was assessed by means of rectal temperature, clinical score, pathological lesion, white blood cell count, viral load, etc.ResultsPTD-poMx1 entered both cell lines within 3 h and maintained for 16 h, but did not affect CSFV binding and uptake. Viral titers and qRT-PCR data showed that PTD-poMx1 inhibited CSFV replication in both cell lines, showing significant antiviral activity after infection. Injection of PTD-poMx1 into CSFV-challenged pigs attenuated CSFV symptoms and viremia in dose-dependent manner but did not completely block virus replication within 14 days post challenge, suggesting that PTD-poMx1 confers partial protection against a lethal challenge.ConclusionWe demonstrated the anti-CSFV activity of PTD-poMx1 in vitro and in vivo. The results have shown that treatment with PTD-poMx1 alleviated symptoms and viral load in infected pigs. The results support our previous in vitro studies and suggest that PTD-poMx1 could be promising in reducing the clinical signs caused by CSFV.

Intracellular transduction of TAT-Hsp27 fusion protein enhancing cell survival and regeneration capacity of cardiac stem cells in acute myocardial infarction

Myocardial infarction (MI) results in the substantial loss of functional cardiomyocytes, which frequently leads to intractable heart disorders. Cardiac stem cells (CSCs) that retain the capacity to replace all cardiac cells might be a promising strategy for providing a source of new functional cardiomyocytes; however, the poor survival and engraftment of transplanted CSCs in the hostile environment of MI critically mitigate their therapeutic benefits. To capitalize their therapeutic potential, an ex vivo strategy in which CSCs were introduced to the recombinant heat shock protein 27 (Hsp27) through a TAT protein transduction domain for increasing the viability and engraftment in the infarcted myocardium was designed. A recombinant TAT fused Hsp27 (TAT-Hsp27) was able to enter CSCs in a dose-dependent manner. CSCs transduced with TAT-Hsp27 expressed not only endogenous Hsp27 but externally introduced Hsp27, resulting in substantial increase of their anti-oxidative and anti-apoptotic properties via suppressing reactive oxygen species production, the MAPKs signaling pathway, and caspase activation. TAT-Hsp27 enabled CSCs to be protected from apoptotic- and hypoxic-induced cell death during in vitro cardiomyogenic differentiation. In vivo studies demonstrated that CSCs transduced TAT-Hsp27 significantly increased the survival and engraftment in the acutely infarcted myocardium, which is closely related to caspase activity suppression. Finally, CSCs transduced TAT-Hsp27 improved cardiac function and attenuated cardiac remodeling in comparison with non-transduced CSCs. Overall, our approach, which is based on the ex vivo intracellular transduction of TAT-Hsp27 into CSCs before myocardial delivery, might be effective in treating MI.

Evidence of in vivo drug delivery via the tat protein transduction domain

Evidence of in vivo drug delivery via the tat protein transduction domain

TAT-OSBP-1-MKK6(E), a novel TAT-fusion protein with high selectivity for human ovarian cancer, exhibits anti-tumor activity.

To improve the selectivity of TAT-fusion proteins for targeted cancer therapy, we developed a novel TAT-based target-specific fusion protein, TAT-OSBP-1-MKK6(E), and evaluated its selectivity and anti-tumor activity in vitro and in vivo. The fusion protein containing TAT-OSBP-1-MKK6(E) has three functional domains: (1) the protein transduction domain of TAT, (2) the human ovarian cancer HO8910 cell-specific binding peptide (OSBP-1) and (3) the potential anti-tumor effector domain of MKK6(E). The transduction efficiency, selectivity, cytotoxicity and apoptotic effect of TAT-OSBP-1-MKK6(E) were examined using immunofluorescence, CCK8 assay and flow cytometry. The in vivo anti-tumor efficacy and target specificity of the fusion protein were evaluated using a nude mouse model with subcutaneous xenografts of human ovarian cancer HO8910 cells. Tumor-bearing mice were divided into three treatment groups that received tail vein injections of TAT-OSBP-1-MKK6(E), TAT-OSBP-1 or normal saline. Tumor growth inhibition was determined by tumor volume, weight and morphology. The distribution and apoptotic effect of TAT-OSBP-1-MKK6(E) were assessed by immunohistochemical staining and TUNEL assays. TAT-OSBP-1-MKK6(E) can be selectively internalized into human ovarian cancer HO8910 cells, rather than normal ovarian OSE cells. In vivo, the fusion protein was mainly expressed in the tumor xenograft, but not in ovary or liver tissues. As a result, TAT-OSBP-1-MKK6(E) significantly induced growth inhibition and apoptosis of tumor cells in vitro and in vivo, with limited effects in normal cells and tissues. TAT-OSBP-1-MKK6(E) treatment can selectively target HO8910 cells in vitro and in vivo, leading to growth inhibition and apoptosis of tumor cells. As such, TAT-OSBP-1-MKK6(E) may be a potential approach for ovarian cancer target therapy.

Transbody against hepatitis B virus core protein inhibits hepatitis B virus replication in vitro.

Hepatitis B virus (HBV) infection is one of the major causes of chronic liver diseases. The current therapeutics show limited efficacy. In the HBV life cycle, virus core antigen (HBcAg) plays important multiple roles. Blocking the pleiotropic functions of HBcAg may thus represent a promising strategy for anti-HBV replication. In this study, monoclonal antibody (MAb) against core antigen of human HBV was coupled with TAT protein transduction domain (TAT PTD) to form transbody, and the effect on virus replication was evaluated in vitro. The HBV transbody, HBcMAb-TAT PTD conjugate, recognized HBcAg and retained cell-penetrating activity in living cells. In HBV-transfected liver cell line HepG2.2.15, HBV transbody suppressed not only the extracellular HBsAg, HBeAg and HBV DNA, but also the intracellular HBsAg, HBeAg, HBcAg and HBV DNA in a dose-dependent manner. These results indicate that the transbody prepared possesses readily cell-penetrating ability and potent antiviral activity, providing a novel approach, a cell-permeable antibody against HBcAg, for the treatment of HBV infection.

Abstract 17773: Peptide Inhibition of PTEN Increases Mouse Survival after Sudden Cardiac Arrest

Introduction: We recently reported that TAT fusion proteins designed to activate versus inhibit the Akt related phosphatase PTEN in heart cells respectively worsen or significantly improve ischemia/reperfusion injury and contractile functional recovery. We therefore designed a short 20 amino acid peptide PTEN inhibitor, TAT-PTEN9c, for rapid tissue delivery and tissue protection after mouse sudden cardiac arrest (SCA). TAT-PTEN9c contains a TAT protein transduction domain fused to the PTEN C-terminal 9 amino acids of the PDZ-domain binding motif. We hypothesized that TAT-PTEN9c blocks PTEN membrane localization and activation, with corresponding tissue Akt activation and improved SCA survival. Methods: Mouse cardiomyocytes were isolated from 1-3 day old mouse pups. Western blot was used to determine the efficacy of TAT-PTEN9c for enhancing Akt phosphorylation in mouse cardiomyocytes exposed to oxidant (H2O2) stress. C57BL6 mice were subjected to an established potassium-induced 8 min SCA protocol. MAP, ETCO2, body temperature and ECG were recorded until 4 h after successful cardiopulmonary resuscitation (CPR). TAT-PTEN9c (7.8 mg/kg) was given intravenously (IV) immediately after CPR (n=10) and a 2nd dose in an additional 5 mice was given at 60 min. IV administration of TAT-GFP was used to measure the kinetics of heart and brain tissue TAT protein delivery. Results: The designed TAT-PTEN9c peptide significantly enhanced Akt activation in neonatal mouse cardiomyoctes in a concentration-dependent manner. 30 mice with successful CPR were randomly assigned to receive either saline or TAT-PTEN9c. Survival (versus cardiovascular collapse and death) was significantly increased in TAT-PTEN9c treated group compared with that of saline control at 2 h (14/15, 93% vs. 9/15, 60%, P &lt; 0.05) and at 4 h (10/15, 67% vs. 6/15, 40%, P &lt; 0.05) after CPR. Western blot and immunohistochemistry showed that TAT protein transduction in heart and brain was diffuse and occurred within 5 min after IV administration. Conclusion: TAT-PTEN9c can be used after CPR in a mouse SCA model to rapidly enhance Akt activation in critical organs and prevent early cardiovascular collapse and death.

Enhanced delivery of human growth hormone across cell membrane by Tat-PTD

In this study, we utilized the HIV protein Tat protein transduction domain (Tat-PTD) to enhance the intestinal absorption of recombinant human growth hormone (rhGH) delivered by oral administration. Insulin-like growth factor 1 (IGF-1), the key factor in the GH signal transduction pathway, was differentially affected at the mRNA level by various concentrations of rhGH. At high rhGH concentrations (500 ng/ml), IGF-1 was downregulated, while low concentrations (5 ng/ml) caused IGF-1 upregulation. The addition of Tat-PTD had a significant facilitating effect on rhGH. Frozen tissue sections visualized with fluorescence microscopy, cultured cells visualized by confocal microscopy and flow cytometry all confirmed that rhGH fused to Tat-PTD demonstrated more intracellular fluorescent signal when compared to rhGH alone. ELISA showed that after 2 h of incubation, Tat-rhGH levels in the rat intestinal cavity were 1.38-fold higher than rhGH. These data indicated that Tat-PTD effectively improved the internalization of rhGH and enhance the signal transduction of rhGH, possibly laying a solid foundation for the novel oral administration of rhGH.

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.

Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.

In vitro inhibition of vesicular stomatitis virus replication by purified porcine Mx1 protein fused to HIV-1 Tat protein transduction domain (PTD)

Vesicular stomatitis virus (VSV) is the causative agent of Vesicular stomatitis (VS), a highly contagious fatal disease of human and pigs. Few effective antiviral drugs are currently available against VSV infection. Mx proteins are interferon (IFN)-induced dynamin-like GTPases present in all vertebrates with a range of antiviral activities. Previous studies have shown that the transfected cell lines expressing either porcine Mx1 or human MxA acquired a high degree of resistance to VSV. To explore the feasibility of taking porcine Mx1 protein expressed in Escherichia coli as an antiviral agent, we applied the pCold system to express this fusion protein (PTD-poMx1), which consisted of an N-terminal HIV-1 Tat protein transduction domain (PTD) and the full-length porcine Mx1, and investigated its effects on the replication of VSV in Vero cells. The results demonstrated that the purified PTD-poMx1 fusion proteins could transduct into cells after incubated for 5h and had no cytotoxic. Furthermore, plaque reduction assay, determination of TCID50, real-time PCR and Western blot analyses were carried out to confirm the antiviral activity of purified fusion proteins in VSV-infected Vero cells. Altogether, these data suggested that PTD-poMx1 fusion proteins might be applicable to inhibit VSV replication as a novel antiviral therapeutic agent.

Fusion of Tat-PTD to the C-terminus of catfish growth hormone enhances its cell uptakes and growth-promoting effects

Fish growth hormone has been long regarded as a promising feed additive in aquaculture for its significant growth promotion effect. To improve its efficiency in oral applications, we generated a Silurus asotus growth hormone-Tat (saGH-Tat) fusion protein and investigated its effect on biological activities relevant to transduction and growth promotion. In this study, saGH, saGH-Tat, Green fluorescent protein and saGH fused protein (GFP-saGH) and GFP-saGH-Tat were all highly expressed in Escherichia coli strain BL21 (DE3) and confirmed by Western blot. The transduction effect of Tat protein transduction domain (Tat-PTD) was verified by observing increased GFP fluorescence in separated intestine cells or frozen sections in GFP-saGH-Tat treated fish. The recombinant saGH and saGH-Tat were tested for biological activity by specific binding with crude liver membrane extracts as well as in a growth promotion assay via oral administration. saGH-Tat treatment demonstrated strengthened receptor binding and accelerated growth-promoting effects. Under optimal concentrations of hormone (1μg/g of fish body weight), saGH-Tat treated fish exhibited an 87.25% growth increase compared to 61.3% in saGH-treated fish. The response of downstream signal pathway factors were analyzed by measuring insulin like growth factor-I (IGF-I), growth hormone receptor (GHR) mRNA expression in the liver, and growth related digestive enzymes (trypsin and chymotrypsin), and all indicate significant up-regulation. Further confirmation of increased GH level in carp blood by Tat fusion was shown by flow cytometry. These results suggested that Tat-PTD fused to the C-terminus saGH greatly enhances its biological activities, making it a promising agent for increasing productivity in aquaculture.