TAT Fusion Proteins Research Articles

Development of Tat-fused drug binding protein to improve anti-cancer effect of mammalian target of rapamycin inhibitors

Development of Tat-fused drug binding protein to improve anti-cancer effect of mammalian target of rapamycin inhibitors

Protein Delivery by PTDs/CPPs.

The ability to deliver or transduce proteins into cells allows for the manipulation of cell biology in culture, preclinical models, and potentially human disease. Fusion proteins containing the TAT peptide transduction domain (PTD), also known as cell-penetrating peptide (CPP), allow for delivery of a wide variety of proteins, including enzymes, transcription factors, tumor suppressor proteins, and many more. TAT-fusion proteins are generated cloning in-frame into the pTAT-HA plasmid, then transformed into E. coli for expression, and purified by the 6-His affinity tag over Ni-NTA column, followed by a final IEX FPLC purification step.

Prokaryotic expression and transmembrane transfer of fusion protein TAT-RIG-I-GFP

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.

Hydrogel-based delivery of Tat-fused protein Hsp70 protects dopaminergic cells in vitro and in a mouse model of Parkinson\u2019s disease

Neurodegenerative disorders such as Parkinson’s disease (PD) have no effective therapies. However, many promising drugs are precluded from clinical trials because of their poor brain availability. The chaperone protein Hsp70 has been reported to be effective in PD models, but its brain targeting is challenging. We developed a novel brain Hsp70 delivery system using injectable, biocompatible, and biodegradable semi-interpenetrating polymer networks of collagen (COLL) and low-molecular-weight hyaluronic acid (LMW HA) structured with gelatin particles. We produced human recombinant Hsp70-1A fused with the cell-penetrating peptide Tat (Tat-Hsp70) that was neuroprotective in vitro against the dopaminergic toxin 6-hydroxydopamine (6-OHDA). We assessed Tat-Hsp70 release from the selected COLL-LMW HA composites in vitro, observing a 95% release of loaded protein after 96 h. The release kinetics FITTED the Korsmeyer-Peppas model (regression coefficient 0.98) and the released Tat-Hsp70 remained neuroprotective for SH-SY5Y cells. Magnetic resonance imaging revealed that COLL-LMW HA composites lasted at least 96 h at the brain level, and in vivo Tat-Hsp70 release studies indicated that hydrogel presence is pivotal for a spatially focused neuroprotective effect. In an in vivo model of dopaminergic degeneration, Tat-Hsp70-loaded composites conveyed neuroprotection at both the behavioral and dopaminergic neuronal levels against the striatal injection of 6-OHDA. After the injection of Tat-Hsp70-loaded composites, mice showed a transient inflammatory response, with a decrease in GFAP and CD11b immunostaining after 7 days. Our delivery system enabled the effective brain release of Tat-Hsp70 and is ready for further improvements.

OBP fused with cell-penetrating peptides promotes liposomal transduction

Cell-penetrating peptides (CPPs) have been applied as novel transport systems with the ability to facilitate the delivery of peptides, proteins, and oligonucleotides into cells. Herein, we designed different fusion proteins composed by pig odorant binding protein (OBP-I) and three CPPs, namely Tat, pVEC and Pep-1. A new methodology using liposomes as reservoirs and OBP:CPPs as carriers was developed as an advanced system to capture odorant molecules. 1-aminoanthracene (1-AMA) was used as a model molecule to evaluate the transduction ability of OBP:CPPs into the reservoirs. The transduction efficiency was dependent on the initial capacity of OBP:CPPs to bind 1-AMA and on the penetration of liposomes promoted by the CPPs. An encapsulation efficiency of 42% was obtained with OBP:Tat fusion protein. The presence of Tat peptide increased the 1-AMA transduction of 1.3 and 2.5 fold compared with Pep-1 and pVEC, respectively. This work expands the application of OBPs and CPPs on the design of promising capture and delivery systems for textile and cosmetic applications.

Human-derived fusogenic peptides for the intracellular delivery of proteins

The cytosolic delivery of therapeutic proteins (e.g., antibodies or enzymes) by cell-penetrating peptides (CPPs), such as a human immunodeficiency virus-derived TAT peptide, is facilitated by fusogenic peptides (FPs). For instance, we recently demonstrated that an FP, B18, which is derived from a sea urchin gamete fusion protein, promotes endosomal escape of an enhanced green fluorescent protein (eGFP)-TAT fusion protein directly conjugated to it. However, the potential clinical use of FPs raises concerns because all conventional FPs are non-human-derived. To solve this problem, we have attempted to identify novel human-derived FPs from two human proteins, including a human sperm protein, IZUMO1, which is involved in gamete recognition and fusion, and a human endogenous retroviral envelope protein, Syncytin1, which is involved in placental morphogenesis. Partial peptides from the core domains of the abovementioned proteins were chosen as candidates to generate human-derived FPs. We prepared fusion proteins of these peptides with eGFP and TAT in Escherichia coli and observed the localization of these fusion proteins in HeLa cells using confocal microscopy. Our results suggested that a 19-residues peptide of Syncytin1 (positions 322–340), named S19, possessed strong intracellular uptake activities with no detectable cytotoxicity. In addition, we estimated the number of molecules that escaped from endosomes using a nuclear localization signal, suggesting that the S19 peptide stimulated the intracellular delivery of TAT-fused eGFP by ~90-fold. Furthermore, we confirmed that S19 promoted the intracellular delivery of eGFP to various human cell lines, including HeLa, A431, HepG2, and SK-N-SH. In addition, we demonstrated that not only eGFP but also SNAP-tag and β-galactosidase were delivered efficiently and retained their activities.

Sumoylation-deficient Prdx6 gains protective function by amplifying enzymatic activity and stability and escapes oxidative stress-induced aberrant Sumoylation

Aberrant Sumoylation of protein(s) in response to oxidative stress or during aging is known to be involved in etiopathogenesis of many diseases. Upon oxidative stress, Peroxiredoxin (Prdx) 6 is aberrantly Sumoylated by Sumo1, resulting in loss of functions and cell death. We identified lysines (K) 122 and 142 as the major Sumo1 conjugation sites in Prdx6. Intriguingly, the mutant Prdx6 K122/142 R (arginine) gained protective efficacy, increasing in abundance and promoting glutathione (GSH) peroxidase and acidic calcium-independent phospholipase A2 (aiPLA2) activities. Using lens epithelial cells derived from targeted inactivation of Prdx6−/− gene and relative enzymatic and stability assays, we discovered dramatic increases in GSH-peroxidase (30%) and aiPLA2 (37%) activities and stability in the K122/142 R mutant, suggesting Sumo1 destabilized Prdx6 integrity. Prdx6−/−LECs with EGFP-Sumo1 transduced or co-expressed with mutant TAT-HA-Prdx6K122/142 R or pGFP-Prdx6K122/142 R were highly resistant to oxidative stress, demonstrating mutant protein escaped and interrupted the Prdx6 aberrant Sumoylation-mediated cell death pathway. Mutational analysis of functional sites showed that both peroxidase and PLA2 active sites were necessary for mutant Prdx6 function, and that Prdx6 phosphorylation (at T177 residue) was essential for optimum PLA2 activity. Our work reveals the involvement of oxidative stress-induced aberrant Sumoylation in dysregulation of Prdx6 function. Mutant Prdx6 at its Sumo1 sites escapes and abates this adverse process by maintaining its integrity and gaining function. We propose that the K122/142R mutant of Prdx6 in the form of a TAT-fusion protein may be an easily applicable intervention for pathobiology of cells related to aberrant Sumoylation signaling in aging or oxidative stress.

Protection and immunological study on two tetraspanin-derived vaccine candidates against schistosomiasis japonicum.

Tetraspanins (TSPs) are proteins found on the surface of helminth parasites of the genus Schistosoma and are regarded as potentially protective antigens. The large extracellular loop of Schistosoma mansoni tetraspanin-2, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. It is well recognized that CD4(+) T-cell-dependent immunity might play an important role against schistosomes; however, the contribution of CD8(+) T cells against multicellular pathogen is still uncertain. The exogenous protein-pulsed dendritic cells (DCs) can easily activate CD4(+) T cells response, while CD8(+) T cells response was relatively difficult to be induced. In this study, we evaluated the immunogenicity of TSP2HD antigen (hydrophilic domain of the S. japonicum tetraspanin-2) and TAT (the protein transduction domain of HIV-1)-coupled TSP2HD protein. As TAT-fused protein could promote major histocompatibility complex class I-dependent antigen presentation in vitro, TAT-TSP2HD-pulsed DCs induced stronger proliferation of schistosome-specific CD8(+) T cells compared with DCs incubated with TSP2HD alone. Vaccination with TAT-TSP2HD-pulsed DCs in vivo could improve disease outcome in S. japonicum-infected mice and was slightly superior to vaccination with DCs treated with TSP2HD. In summary, these data showed that TAT fusion proteins could help activate CD8(+) cells and Th1 cells and provide part protection against schistosome.

Tat-NOL3 protects against hippocampal neuronal cell death induced by oxidative stress through the regulation of apoptotic pathways.

Oxidative stress-induced apoptosis is associated with neuronal cell death and ischemia. The NOL3 [nucleolar protein3 (apoptosis repressor with CARD domain)] protein protects against oxidative stress-induced cell death. However, the protective mechanism responsible for this effect as well as the effects of NOL3 against oxidative stress in ischemia remain unclear. Thus, we examined the protective effects of NOL3 protein on hydrogen peroxide(H2O2)-induced oxidative stress and the mechanism responsible for these effects in hippocampal neuronal HT22 cells and in an animal model of forebrain ischemia using Tat-fused NOL3 protein(Tat-NOL3). Purified Tat-NOL3 protein transduced into the H2O2-exposed HT22 cells and inhibited the production of reactive oxygen species(ROS), DNA fragmentation and reduced mitochondrial membrane potential(ΔΨm). In addition, Tat-NOL3 prevented neuronal cell death through the regulation of apoptotic signaling pathways including Bax, Bcl-2, caspase-2, -3and-8, PARP and p53. In addition, Tat-NOL3 protein transduced into the animal brains and significantly protected against neuronal cell death in the CA1 region of the hippocampus by regulating the activation of microglia and astrocytes. Taken together, these findings demonstrate that Tat-NOL3 protein protects against oxidative stress-induced neuronal cell death by regulating oxidative stress and by acting as an anti-apoptotic protein. Thus, we suggest that Tat-NOL3 represents a potential therapeutic agent for protection against ischemic brain injury.

The Tyrosine Kinase Pyk2 Contributes to Complement-Mediated Phagocytosis in Murine Macrophages

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family and is mainly expressed in neuronal and hematopoietic cells. As FAK family members are involved in signaling connections downstream of integrins, we studied the role of Pyk2 in complement-receptor 3 (CR3, also known as Mac-1, integrin α_Mβ₂, CD11b/CD18)-mediated phagocytosis, a key process in innate immunity. Using 3 independent approaches, we observed that Pyk2 contributes to CR3-dependent phagocytosis by RAW 264.7 macrophages, but is dispensable for Fcγ receptor (FcγR)-mediated uptake. Reduction of Pyk2 expression levels via siRNA, the pharmacological inhibition of Pyk2 kinase activity as well as macrophage treatment with a cell permeable TAT fusion protein containing the C-terminus of Pyk2 (TAT-PRNK) significantly impaired CR3-mediated phagocytosis without affecting FcγR-mediated uptake. In addition, Pyk2 was strongly recruited to complement opsonized Escherichia coli and the pharmacological inhibition of Pyk2 significantly decreased uptake of the bacteria. Finally, CRISPR/Cas-mediated disruption of the pyk2 gene in RAW 264.7 macrophages confirmed the role of this protein tyrosine kinase in CR3-mediated phagocytosis. Together, our data demonstrate that Pyk2 selectively contributes to the coordination of phagocytosis-promoting signals downstream of CR3, but is dispensable for FcγR-mediated phagocytosis.

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.

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 < 0.05) and at 4 h (10/15, 67% vs. 6/15, 40%, P < 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.

LIFRα-CT3 induces differentiation of a human acute myelogenous leukemia cell line HL-60 by suppressing miR-155 expression through the JAK/STAT Pathway

The distal cytoplasmic motifs of the leukemia inhibitory factor receptor α-chain (LIFRα-CT3) and its TAT fusion protein (TAT-CT3) can independently suppress cell viability and induce myeloid differentiation in human leukemia HL-60 cells in our previous studies. But its underlying mechanism remains undefined. Herein, we show that a prokaryotic expressed TAT-CT3 induced a rapid elevation of STAT3 phosphorylation (pSTAT3), and then suppress the transcription of miR-155 and induce the elevation of SOCS-1, which further inhibited STAT3 phosphorylation for a long-term period. Our result indicated a novel mechanism of TAT-CT3 to promote HL60 cells differentiation, which provides some potential therapeutic targets for future acute myelogenous leukemia therapy.

TAT-protein blockade during ischemia/reperfusion reveals critical role for p85 PI3K-PTEN interaction in cardiomyocyte injury.

Recent work shows that cooling protection after mouse cardiac arrest and cardiomyocyte ischemia is mediated by Akt activation. The PI3K p85 subunit can either augment or inhibit Akt activation depending on its binding to p110 or PTEN respectively. To further clarify the role of PI3K p85 in cardioprotection, we studied novel TAT-p85 fusion proteins that selectively inhibit PI3K p85 binding. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would decrease Akt phosphorylation and abrogate cardioprotection. Microscopy and Western blot analysis demonstrated that TAT fusion protein was transduced into cardiomyocytes within 5 min and remained more than 2 h. Inhibition of PI3K/Akt by TAT-Δp110 p85 significantly increased cell death from 44.6±2.7% to 92.5±3.4% after simulated ischemia and reperfusion. By contrast, PTEN inhibition using TAT-ΔPTEN p85 decreased cell death to 11.9±5.3%, a similar level of cardioprotection seen with past cooling studies. Additional studies with the small molecule PTEN inhibitor VO-OHpic confirmed that PTEN inhibition was highly protective against cell death induced by ischemia and reperfusion. We conclude that blockade of p85-PTEN interaction and PTEN inhibition may be promising strategies for rescuing the heart from ischemia and reperfusion injury.

Abstract 18534: TAT-Protein Blockade of PTEN After Cardiac Ischemia is Highly Protective

Introduction: Cooling protects against cardiac ischemia/reperfusion (I/R) injury by enhancing PI3K/Akt/NOS signaling. PI3K/Akt signaling depends on binding by its regulatory subunit p85 to the catalytic subunit p110 or the inhibitory phosphatase PTEN. We studied novel TAT-p85 fusion proteins that selectively inhibit these competing p85 interactions. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection in a heart cell model of I/R. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would abrogate protection. To further test TAT fusion proteins for the I/R injury of mouse cardiac arrest (CA), we studied whether a TAT-fused PTEN C-terminal 9 amino acid (TAT-PTEN9c) PTEN inhibitor would activate Akt in critical organs and improve survival. Methods: A TAT-Δp110 p85 plasmid was constructed with nucleotide deletions at amino acids 478-513. The TAT-ΔPTEN p85 plasmid deleted amino acids 1-313. TAT-PTEN9c was synthesized by Anaspec. Cardiomyocytes were isolated from 1-2-day old C57BL6/J mice and exposed to up to 90 min ischemic arrest with 3 h reperfusion (I/R). Cell viability was evaluated by propidium iodide uptake and p-Akt was measured by Western blot. C57BL6 mice were subjected to an established potassium-induced CA protocol. TAT-PTEN9c (7.8 mg/kg) was given IV after return of spontaneous circulation (ROSC) (n=10) and the 2nd dose for another 5 mice was given at 60 min after ROSC. Results: TAT-Δp110 p85 increased cell death from 44.6 +/- 2.7% to 92.5 +/- 3.4%, while TAT-ΔPTEN p85 decreased cell death caused by I/R from 47.8 +/- 1.4% to 11.9 +/- 5.3%. 30 mice were randomized to either saline or TAT-PTEN9c post-ROSC. TAT fusion proteins could be seen within heart and brain tissues within 2 minutes of IV infusion. The survival rate was significantly increased in TAT-PTEN9c treated mice compared with that of saline treated mice at 2 hrs (14/15, 93% vs. 9/15, 60%, P < 0.05) and at 4 hrs (10/15, 67% vs. 6/15, 40%, P < 0.05) after ROSC. Conclusions: TAT fusion protein inhibition of PTEN during cardiac I/R and after cardiac arrest appear to be promising strategies for enhancing Akt activation and survival.

Inhibition of Neutrophil Exocytosis Ameliorates Acute Lung Injury in Rats

Exocytosis of neutrophil granules contributes to acute lung injury (ALI) induced by infection or inflammation, suggesting that inhibition of neutrophil exocytosis in vivo could be a viable therapeutic strategy. This study was conducted to determine the effect of a cell-permeable fusion protein that inhibits neutrophil exocytosis (TAT-SNAP-23) on ALI using an immune complex deposition model in rats. The effect of inhibition of neutrophil exocytosis by intravenous administration of TAT-SNAP-23 on ALI was assessed by albumin leakage, neutrophil infiltration, lung histology, and proteomic analysis of bronchoalveolar lavage fluid (BALF). Administration of TAT-SNAP-23, but not TAT-control, significantly reduced albumin leakage, total protein levels in the BALF, and intra-alveolar edema and hemorrhage. Evidence that TAT-SNAP-23 inhibits neutrophil exocytosis included a reduction in plasma membrane CD18 expression by BALF neutrophils and a decrease in neutrophil granule proteins in BALF. Similar degree of neutrophil accumulation in the lungs and/or BALF suggests that TAT-SNAP-23 did not alter vascular endothelial cell function. Proteomic analysis of BALF revealed that components of the complement and coagulation pathways were significantly reduced in BALF from TAT-SNAP-23-treated animals. Our results indicate that administration of a TAT-fusion protein that inhibits neutrophil exocytosis reduces in vivo ALI. Targeting neutrophil exocytosis is a potential therapeutic strategy to ameliorate ALI.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

New Novel Bone Fusion Using TAT-BMP Hydrogel in Rat Model

Introduction Despite bone fusion using BMP is promising, its clinical applications are limited due to high cost and unwanted ectopic bony fusion. Furthermore, its processing is complicated and low yield. Recently, new method of BMP producing has been developed using TAT protein domain, which can facilitate the passage of high molecular protein into the cell. Thus BMP precursor with TAT can pass the cell membrane and convert into BMP after chemical degradation. It can secret as BMP itself and activate intrinsic BMP production. Basic animal studies showed rapid diffusion of TAT-BMP in spite of carrier material. For stable release of TAT-BMP, Chitosan hydrogel was made with TAT-BMP and applied to rat model. Materials and Methods Under intraperitoneal anesthesia, the rat thoracolumbar spine was exposed as usual spinal procedure, 1–2 spinous processes were removed and TAT-BMP hydrogel (total 1 cc in volume) or Biopad (calcium bisphosphte) was implanted and operative wound was closed. Total 12 rats randomly assign for TAT-BMP hydrogel (8 rats) and Biopad (4 rats). Animals were survived for 2 months and sacrificed for radiologic and histological examination. Simple X-ray and micro-CT were taken, and histological examination (H & E staining) was done. Results On gross examination, all TAT-BMP hydrogel groups showed palpable mass, and it confirmed with histological finding. Unfortunately, mature bone formation was not even due to short survival period. There is insufficient release of hydrogel in some cases, but there were no ectopic bone formation. On micro-CT, bone trabeculation was visible, but not enough like mature bone. Conclusion Although it was preliminary results, bone fusion using TAT-BMP hydrogel was promising. Further studies using bigger animal and titration of hydrogel with longer survival period are mandatory for clinical implementation. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Regulation of hematopoietic stem cells using protein transduction domain–fused Polycomb

The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.

TAT-Mediated Delivery of p27 in Tumor Cell Lines as a Potential Therapeutic Peptide

TAT-fusion proteins (TAT-wt-p27, TAT-pt-p27, TAT-N'-p27) were introduced into the cells by protein transduction method. The mechanism by which transduced 27 influences on the regulation of cell cycle and apoptosis, were explored. TAT-p27-fusion proteins affected the proliferation of examined cell lines depending on type of the cells and protein. Transduced p27 induced accumulation of cyclin D1 and D3, with slight differences among the form of protein. Expression of cyclin D2 and E was mainly unchanged. Furthermore, TAT fusion proteins promoted apoptosis, which resulted in activation of caspase 3, appearance of poly (ADP-ribose) polymerase and DNA fragments, as well as the activation of apoptosis-inducing factor. The results pointed that transduced p27 activates apoptosis through activation of different signal transduction pathways. Thus, the molecule of p27 could be appropriate for treatment of tumors with deregulated its function. Also, the protein transduction method could find the application in specifically targeted cancer therapy.