Functional expression and purification of recombinant full-length human ATG7 protein with HIV-1 Tat peptide in Escherichia coli.

The human autophagy-related protein ATG7 (hATG7), an E1-like ubiquitin enzyme, activates two ubiquitin-like proteins, LC3 (Atg8) and Atg12, and promotes autophagosome formation. While hATG7 plays an essential role for the autophagy conjugation system, the production of full-length functional hATG7 in bacterial systems remains challenging. Previous studies have demonstrated that the HIV-1 virus-encoded Tat peptide ('GRKKRRQRRR') can increase the yield and solubility of heterologous proteins. Here, functional full-length hATG7 was expressed using the pET28b-Tat expression vector in the Escherichia coli BL21 (DE3) strain. Recombinant hATG7 protein aggregated as inclusion bodies while expressed with widely used prokaryotic expression plasmids. In contrast, the solubility of Tat-tagged hATG7 increased significantly with prolonged time compared to Tat-free hATG7. The recombinant proteins were purified to >90% homogeneity under native conditions with a single step of affinity chromatography purification. The results of in vitro pull-down and LC3B-I lipidation assays showed that Tat-tagged hATG7 directly interacted with LC3B-I and promoted LC3B-I lipidation, suggesting that Tat-tagged hATG7 has significant catalytic activity. Overall, this study provides a novel method for improving the functional expression of full-length hATG7 in bacterial systems by fusion with the Tat peptide, a process which may be applied in future studies of hATG7 structure and function.

Production and Transduction of a Human Recombinant ß-Globin Chain into Proerythroid K-562 Cells To Replace Missing Endogenous ß-Globin.

Protein replacement therapy (PRT) has been applied to treat severe monogenetic/metabolic disorders characterized by a protein deficiency. In disorders where an intracellular protein is missing, PRT is not easily feasible due to the inability of proteins to cross the cell membrane. Instead, gene therapy has been applied, although still with limited success. ß-Thalassemias are severe congenital hemoglobinopathies, characterized by deficiency or reduced production of the adult ß-globin chain. The resulting imbalance of α-/ß-globin chains of adult hemoglobin (α2ß2) leads to precipitation of unpaired α-globin chains and, eventually, to defective erythropoiesis. Since protein transduction domain (PTD) technology has emerged as a promising therapeutic approach, we produced a human recombinant ß-globin chain in fusion with the TAT peptide and successfully transduced it into human proerythroid K-562 cells, deficient in mature ß-globin chain. Notably, the produced human recombinant ß-globin chain without the TAT peptide, used as internal negative control, failed to be transduced into K-562 cells under similar conditions. In silico studies complemented by SDS-PAGE, Western blotting, co-immunoprecipitation and LC-MS/MS analysis indicated that the transduced recombinant fusion TAT-ß-globin protein interacts with the endogenous native α-like globins to form hemoglobin α2ß2-like tetramers to a limited extent. Our findings provide evidence that recombinant TAT-ß-globin is transmissible into proerythroid K-562 cells and can be potentially considered as an alternative protein therapeutic approach for ß-thalassemias.

Expression and purification of TAT-NDRG2 recombinant protein and evaluation of its anti-proliferative effect on LNCaP cell line.

N-myc downstream regulated gene2 (NDRG2) belongs to tumor suppressor protein family of NDRG. Anti-proliferative and anti-metastasis of NDRG2 overexpression has been demonstrated in a number of tumors. The aim of this study was to fuse the gene of Trans Activator of Transcription (TAT) protein transduction domain with NDRG2 gene and express and purify TAT-NDRG2 fusion protein in order to investigate the effects of TAT-NDRG2 protein on proliferation and apoptosis of LNCaP prostate carcinoma cell line. pET28a-TAT-NDRG2 and pET28a-NDRG2 plasmids were constructed and transformed into E. coli-BL21(DE3). TAT-NDRG2 and NDRG2 proteins were expressed in the bacteria, purified using affinity chromatography and verified using western blotting. The effects of TAT-NDRG2 and NDRG2 protein treatment on LNCaP cells proliferation and apoptosis were evaluated using MTT assay and AnnexinV, 7-AAD flow cytometry assay, respectively. Western blot analysis confirmed the expression and purification of TAT-NDRG2 and NDRG2 proteins. Treatment of LNCaP cells with TAT-NDRG2 protein increased cell death and induced apoptosis significantly (P < 0.05) compared to control and NDRG2 protein-treated cells. These results suggest that TAT-NDRG2 protein can be considered as a therapeutic modality for the treatment of tumors.

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress.

HIV-1 Tat biosensor: Current development and trends for early detection strategies.

Human immunodeficiency virus (HIV) has infected almost 35 million people worldwide. Various tests have been developed to detect the presence of HIV during the early stages of the disease in order to reduce the risk of transmission to other humans. The HIV-1 Tat protein is one of the proteins present in HIV that are released abundantly approximately 2-4 weeks after infection. In this review, we have outlined various strategies for detecting the Tat protein, which helps transcribe the virus and enhances replication. Detection strategies presented include immunoassays, biosensors and gene expression, which utilize antibodies or aptamers as common probes to sense the presence of Tat. Alternatively, measuring the levels of gene transcription is a direct method of analysing the HIV gene to confirm the presence of Tat. By detection of the Tat protein, virus transmission can be detected in high-risk individuals in the early stages of the disease to reduce the risk of an HIV pandemic.

HIV-1 Tat C modulates NOX2 and NOX4 expressions through miR-17 in a human microglial cell line.

HIV-1 invades CNS in the early course of infection, which can lead to the cascade of neuroinflammation. NADPH oxidases (NOXs) are the major producers of reactive oxygen species (ROS), which play important roles during pathogenic insults. The molecular mechanism of ROS generation via microRNA-mediated pathway in human microglial cells in response to HIV-1 Tat protein has been demonstrated in this study. Over-expression and knockdown of microRNAs, luciferase reporter assay, and site-directed mutagenesis are main molecular techniques used in this study. A significant reduction in miR-17 levels and increased NOX2, NOX4 expression levels along with ROS production were observed in human microglial cells upon HIV-1 Tat C exposure. The validation of NOX2 and NOX4 as direct targets of miR-17 was done by luciferase reporter assay. The over-expression and knockdown of miR-17 in human microglial cells showed the direct role of miR-17 in regulation of NOX2, NOX4 expression and intracellular ROS generation. We demonstrated the regulatory role of cellular miR-17 in ROS generation through over-expression and knockdown of miR-17 in human microglial cells exposed to HIV-1 Tat C protein. Activated microglial cells mediated neuroinflammatory events are observed in HIV-associated neurological disorders. The reduction in miR-17 levels was observed in microglial cells exposed to HIV-1 Tat C protein. miR-17 regulated the expression of NOX2 and NOX4, which in turn regulated the reactive oxygen species (ROS) production in microglial cells. Increased ROS production led to the activation of microglial cells and increased cytokine production. This study thus demonstrated a novel miR-17-mediated regulatory pathway of ROS production in microglial cells. HMC3 = human microglia clone 3 cell lines.

Expression and purification of human TAT-p53 fusion protein in Pichia pastoris and its influence on HepG2 cell apoptosis.

P53 is an attractive target in molecular cancer therapeutics because of its critical role in regulating cell cycle arrest and apoptosis. The limitations in the development of p53-based cancer therapeutic strategy include its inefficient transmission through cell membrane of tumor cells and low protein yields in the expression system. In the present study, p53 was fused with HIV TAT protein, which can cross cell membranes, and expressed by Pichia pastoris. Stable production of Tat-p53 was achieved. After being transduced with Tat-p53 protein, the growth of cancer cell line, HepG2, was inhibited by increased apoptosis in culture. This expression system could thus be utilized to produce human Tat-p53 fusion protein.

A designed Tat immunogen generates enhanced anti-Tat C-terminal antibodies.

HIV-1 Tat has been identified as an attractive target for vaccine development and is currently under investigation in clinical trials as both a therapeutic and preventative vaccine for HIV-1. The Tat C-terminal region is of significant importance for its extracellular activity. In this study, we designed two recombinant Tat immunogens, Tat(B41-100N) and Tat(B41-100C), with two extended Tat C-terminal regions (41-100 aa) and compared their humoral immune response with native Tat. Interestingly, our results showed that Tat(B41-100C) elicited a higher antibody titer than Tat and Tat(B41-100N) in both mice and rabbits. The recombinant fusion protein-based epitope analysis showed that Tat(B41-100C) induced a remarkably enhanced humoral immune response against extended Tat C-terminal regions containing residues 38-100, 49-100 and 60-100. Our study demonstrates that the designed Tat(B41-100C) presents a designed immunogenicity that elicits enhanced Tat-specific antibodies especially against extended Tat C-terminal regions.

TAT-mediated delivery of Tousled protein to salivary glands protects against radiation-induced hypofunction.

PURPOSE: Patients treated with radiotherapy for head-and-neck cancer invariably suffer its deleterious side effect, xerostomia. Salivary hypofunction ensuing from the irreversible destruction of glands is the most common and debilitating oral complication affecting patients undergoing regional radiotherapy. Given that the current management of xerostomia is palliative and ineffective, efforts are now directed toward preventive measures to preserve gland function. The human homolog of Tousled protein, TLK1B, facilitates chromatin remodeling at DNA repair sites and improves cell survival against ionizing radiation (IR). Therefore, we wanted to determine whether a direct transfer of TLK1B protein to rat salivary glands could protect against IR-induced salivary hypofunction. METHODS: The cell-permeable TAT-TLK1B fusion protein was generated. Rat acinar cell line and rat salivary glands were pretreated with TAT peptide or TAT-TLK1B before IR. The acinar cell survival in vitro and salivary function in vivo were assessed after radiation. RESULTS: We demonstrated that rat acinar cells transduced with TAT-TLK1B were more resistant to radiation (D0 = 4.13 ± 1.0 Gy; α/ß = 0 Gy) compared with cells transduced with the TAT peptide (D0 = 4.91 ± 1.0 Gy; α/ß = 20.2 Gy). Correspondingly, retroductal instillation of TAT-TLK1B in rat submandibular glands better preserved salivary flow after IR (89%) compared with animals pretreated with Opti-MEM or TAT peptide (31% and 39%, respectively; p < 0.01). CONCLUSIONS: The results demonstrate that a direct transfer of TLK1B protein to the salivary glands effectively attenuates radiation-mediated gland dysfunction. Prophylactic TLK1B-protein therapy could benefit patients undergoing radiotherapy for head-and-neck cancer.

Purification of TAT-CC-HA protein under native condition, and its transduction analysis and biological effects on BCR-ABL positive cells.

BCR-ABL oncoprotein is the cause of chronic myeloid leukemia. The homologous oligomerization of BCR-ABL protein mediated by BCR coiled-coil (CC) domain plays an important role in ABL kinase activation. The HIV-1 TAT peptide has been used extensively for the introduction of proteins into cells. We recombinated a TAT-CC-HA protein to interrupt the homologous oligomerization of BCR-ABL. The expression conditions for TAT-CC-HA were optimized. The TAT-CC-HA fusion protein was purified with Ni+-NTA resin. TAT-CC-HA fusion protein was added into the cultures of Ba/F3-p210, 32D-p210, K562, KU812, Ba/F3, 32D, and HL-60 cells. It was found that TAT-CC-HA could transduce into these cells. It was confirmed that TAT-CC-HA fusion protein was internalized by Ba/F3-p210, K562, and Ba/F3 cells and located in the cytoplasm observed by confocal laser scanning fluorescence microscope. The transduction of TAT-CC-HA fusion protein into K562 cells was in a dose-dependent and time-dependent manner. The result of coimmunoprecipitation assay indicated that TAT-CC-HA could interact with BCR-ABL in K562 cells. The effects of TAT-CC-HA fusion protein on cell growth and apoptosis were detected by MTT test and flow cytometry. Our findings suggested that TAT-CC-HA fusion protein could specifically inhibit the growth of BCR-ABL positive cells, and specifically induce apoptosis of BCR-ABL positive cells, while not affect the growth and apoptosis of BCR-ABL negative cells.

Matrix metalloproteinase-1 induces cleavage of exogenous alphaB-crystallin transduced by a cell-penetrating peptide.

Cell-penetrating peptides (CPPs), including TAT-CPP, have been used to deliver exogenous proteins into living cells. Although a number of proteins fused to TAT-CPP can be delivered into various cells, little is known about the proteolytic cleavage of TAT-fusion proteins in cells. In this study, we demonstrate that a small heat shock protein (sHSP), alphaB-crystallin (αB-crystallin), delivered by TAT-CPP is susceptible to proteolytic cleavage by matrix metalloproteinase-1 (MMP-1) in cardiac myoblast H9c2 cells. Recombinant TAT-αB-crystallin was efficiently transduced into H9c2 cells. For a few hours following protein transduction, generation of a 14-kDa fragment, a cleavage band of TAT-αB-crystallin, increased in a time-dependent manner. This fragment was observed only in detergent-insoluble fractions. Interestingly, treatment with MMP inhibitors blocked the cleavage of TAT-αB-crystallin. In test tubes, recombinant MMP-1 processed TAT-αB-crystallin to generate the major cleavage fragment 14-kDa, as observed in the cells treated with TAT-αB-crystallin. The N-terminal sequences of the 14-kDa fragment were identified as Leu-Arg-Ala-Pro-Ser-Trp-Phe, indicating that this fragment is generated by cleavage at Phe54-Leu55 of αB-crystallin. The MMP-1-selective inhibitor abolished the production of 14-kDa fragments in cells. In addition, the cleaved fragment of TAT-αB-crystallin was significantly reduced in cells transfected with MMP-1 siRNA. Moreover, the enzymatic activity of MMP-1 was markedly increased in TAT-αB-crystallin-treated cells. TAT-αB-crystallin has a cytoprotective effect on H9c2 cells under hypoxic insult, moreover, MMP-1-selective inhibitor treatment led to even increased cell viability. These results suggest that MMP-1 is responsible for proteolytic cleavage of TAT-αB-crystallin during its intracellular transduction in H9c2 cells.

Recombinant human TAT-OP1 to enhance NGF neurogenic potential: preliminary studies on PC12 cells.

Osteogenic protein 1 (OP1), also known as bone morphogenic protein-7 (BMP7), is a multifunctional cytokine with demonstrated neurogenic potential. As the recombinant OP1 (rhOP1) was shown to provide axonal guidance cues and to prevent the reduction of dendritic growth in the injury-induced cortical cultures, it was suggested that an in vivo efficient rhOP1 delivery could enhance neurite growth and functional reconnectivity in the damaged brain. In the present work, we engineered a chimeric molecule in which rhBMP7 was fused to a protein transduction domain derived from HIV-1 TAT protein to deliver the denatured recombinant BMP7 into cells and obtain its chaperone-mediated folding, circumventing the expensive and not much efficient in vitro refolding procedures. When tested on rat PC12 cells, a widely used in vitro neurogenic differentiation model, the resulting fusion protein (rhTAT-OP1) demonstrated to enter fastly into the cells, lose HIV-TAT sequence and interact with membrane receptors activating BMP pathway by SMAD 1/5/8 phosphorylation. In comparison with nerve growth factor (NGF) and BMP7, it proved itself effective to induce the formation of more organized H and M neurofilaments. Moreover, if used in combination with NGF, it stimulated a significant (P < 0.05) and more precocious dendritic outgrowth with respect to NGF alone. These results indicate that rhTAT-OP1 fused with TAT transduction domain shows neurogenic activity and may be a promising enhancer factor in NGF-based therapies.

Cellular siRNA delivery using TatU1A and photo-induced RNA interference.

RNA interference (RNAi)-mediated silencing of specific genes represents a powerful tool for analyzing protein function. It also has profound biotechnological applications for cellular engineering and therapeutics. However, it is necessary to have a method that controls RNAi in response to artificially regulated stimulation. We designed a fluorescently labeled carrier protein to deliver short hairpin RNA (shRNA) with activity that could be regulated via photostimulation. We constructed a cell-permeable RNA-binding protein (RBP) by fusing the U1A RBP and a HIV-1 Tat peptide, which was labeled with an Alexa Fluor 546 fluorophore (TatU1A-Alexa). TatU1A-Alexa bound specifically to shRNA, which contains a U1A-binding sequence. The TatU1A-Alexa/shRNA complex was then internalized into cells via an endocytotic pathway and redistributed from endosomes to the cytosol by photostimulation, which induced RNAi-mediated gene silencing. This successive strategy was termed CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi).

High yield expression and purification of HIV-1 Tat1-72 for structural studies.

The HIV-1 transactivator of transcription (Tat) is a protein essential for virus replication. Tat is an intrinsically disordered RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven Cys residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of 6xHistidine-tagged and isotopically enriched (in N15 and C13) recombinant HIV-1 Tat(1-72) (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the backbone NMR resonances. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced, monomeric, and unfolded in aqueous solution at pH 4.

Tat-modified leptin is more accessible to hypothalamus through brain-blood barrier with a significant inhibition of body-weight gain in high-fat-diet fed mice.

Obesity in human was found mainly due to the poor transportation of leptin through brain-blood barrier (BBB), called as leptin resistance. To produce a leptin capable of penetrating BBB, we have added Tat-PTD(9) to the C terminal of leptin to construct a fusion protein. The fusion Tat-leptin and native leptin genes were synthesized by single-step insertion of a polymerase chain reaction and expressed in Escherichia coli BL21 (Rosseta). The expressing products were purified and renatured by Ni-NTA affinity chromatography, and identified by the molecular size in SDS-PAGE gel and by its immunoreactivity to specific antibody with Western-blotting assay. To bio-functionally evaluate the fusion protein, Balb/c mice fed with high-fat diet (HFD) were given Tat-leptin, leptin or saline for 19 days. The immunohistochemical staining showed the increases in positive stains for the leptin in the region of hypothalamus of the HFD mice with either Tat-leptin or leptin as compared to saline group, but the staining intensity and frequency in the group with Tat-leptin were stronger and higher than those in the group with leptin. Furthermore, the most efficiency in preventing the body-weight gain caused by HFD was found in Tat-leptin group among these three groups. These results suggest that Tat-modified leptin may become a great potential candidate for the prevention or therapy of obese patients.

[Expression and purification of TAT-HBX-EGFP fusion protein and its transmembrane distribution in mouse].

OBJECTIVE: To highly express TAT-HBX-EGFP fusion protein and study its distribution in mouse liver. METHODS: TAT-HBX-EGFP recombinant vector was constructed and fusion protein was induced by IPTG and expression in BL21; fusion protein was purified by Ni-NTA argarose, then injected into the peritoneal cavity of the mice. Distribution of fusion protein was observed by immunofluorescence. RESULTS: TAT-HBX-EGFP was highly expression in E. coli; HBX could be induced into mouse liver by TAT. CONCLUSION: HBX protein could be induced into mouse liver by TAT induced peptide.

HIV-1 Tat-mediated protein transduction of human brain creatine kinase into PC12 cells.

Epilepsy is characterized by the presence of spontaneous episodes of abnormal neuronal discharges and its pathogenic mechanisms remain poorly understood. Recently, we found that the expression of creatine kinase (CK) was markedly decreased in an epilepsy animal model using proteomic analysis. A human CK gene was fused with a HIV-1 Tat peptide to generate an in-frame Tat-CK fusion protein. The purified Tat-CK fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-CK fusion protein was stable for 48 h. Moreover, the Tat-CK fusion protein markedly increased endogenous CK activity levels within the cells. These results suggest that Tat-CK provides a strategy for the therapeutic delivery of proteins in various human diseases including the delivery of CK for potential epilepsy treatment.

TAT-Hsp40 inhibits oxidative stress-mediated cytotoxicity via the inhibition of Hsp70 ubiquitination.

Heat shock protein 40 (Hsp40) functions as a co-chaperone of mammalian Heat shock protein 70 (Hsp70) and facilitates the ATPase activity of Hsp70, and also promotes the cellular protein folding and renaturation of misfolded proteins. In an effort to assess the effects of Hsp40, we generated TAT-fused Hsp40 (TAT-Hsp40). The cells were transduced with TAT-Hsp40 and exposed to H(2)O(2). We demonstrated that the TAT-Hsp40-transduced cells were more resistant to cellular cytotoxicity and cell death. In particular, the degradation of Hsp70 was significantly reduced in TAT-Hsp40-containing cells as a consequence of reduced ubiquitin-proteasome activity after oxidative injury. These data support the notion that Hsp40 may confer resistance to oxidative stress via the prevention of proteasome activity.

The Tat protein broadens T cell responses directed to the HIV-1 antigens Gag and Env: implications for the design of new vaccination strategies against AIDS.

We have previously shown that the biologically active Tat protein targets and efficiently enters dendritic cells, and increases the proteolytic activities of the immunoproteasome, thereby favoring the generation and presentation of the subdominant MHC-I binding CTL epitopes of heterologous antigens. In the present study, we demonstrate that Tat broadens in vivo epitope-specific T cell responses directed to heterologous antigens including HIV structural proteins. Specifically, co-immunization of mice with OVA and Tat proteins induces CTL responses against subdominant and cryptic OVA-derived epitopes, which are not detected in mice vaccinated with OVA alone. Similarly, mice vaccinated with the HIV-1 Gag, Env or V2-deleted Env antigens in combination with Tat show Th1-type and CTL responses directed to a larger number of T cell epitopes, as compared to mice vaccinated with these proteins in absence of Tat. In contrast, Tat did not affect Th2-type responses to these structural HIV proteins. These results indicate that Tat is not only an antigen but also a novel Th1-type adjuvant capable of broadening in vivo the spectrum of epitopes recognized by T cells, and suggest that Tat can be considered an optimal co-antigen in the development of novel vaccination strategies against AIDS.