RESUMO
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) belongs to the TNF cytokine superfamily that specifically induces apoptosis in a broad spectrum of human cancer cell lines but not in most healthy cells. The antitumor potential of recombinant human TRAIL (rhTRAIL) has attracted great attention among biologists and oncologists. However, attempts to express rhTRAIL in Escherichia coli often results in limited yield of bioactive protein due to the formation of inclusion bodies (IBs), which are dense insoluble particulate protein aggregates inside cells. We describe herein a highly simplified method to produce pure bioactive rhTRAIL using E. coli. The method is straightforward and requires only basic laboratory equipment, with highly efficient purification and high yield of renaturation, and may also be applied to produce other proteins that form IBs in E. coli.
Assuntos
Proteínas Recombinantes de Fusão , Ligante Indutor de Apoptose Relacionado a TNF , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia de Afinidade , Cromatografia em Gel , Escherichia coli/metabolismo , Humanos , Corpos de Inclusão/química , Redobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
The cytokine TRAIL induces apoptosis in tumor cells of various origin without affecting normal cells. Clinical trials of TRAIL-receptor (DR4 and DR5) agonists (recombinant TRAIL or death receptors antibodies) have largely failed because most human tumors were resistant to them. Currently, a second generation of agents targeted at TRAIL-R with increased efficiency has been developed. To this end, we have developed DR5-B, a variant of TRAIL selectively interacting with DR5. We have developed a new efficient method for production of TRAIL and DR5-B using expression of these proteins in Escherichia coli strain SHuffle B. The proteins were isolated from the cytoplasmic fraction of cells and purified to a high degree of homogeneity using metal-affinity and ion-exchange chromatography. The protein yield was 211 and 173 mg from one liter of cell culture for DR5-B and TRAIL, respectively, which significantly exceeded the results obtained by other methods. DR5-B killed tumor cells of different origin more efficiently and rapidly compared with TRAIL. The resulting preparations can be used for the study of TRAIL signaling pathways and in preclinical and clinical trials as antitumor agents.
Assuntos
Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Escherichia coli is an essential host for large-scale expression of heterologous polypeptides. However, further applications are limited by the formation of potential protein aggregates. In this work, we developed a novel on-column tag removal and purification system based on Fh8 hydrophobic interaction chromatography purification and ΔI-CM self-cleavage to obtain soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We evaluated several methods to improve TRAIL solubility and finally demonstrated that the Fh8 tag was a powerful solubility enhancer. Finally, we replaced the tobacco etch virus (TEV) protease site with a ΔI-CM self-cleavage intein to simplify the purification process. The released soluble TRAIL purity and yield reached 98.4% and 82.1â¯mg/L in shake flasks, respectively. Thus, the Fh8-ΔI-CM system enhanced target protein solubility by Fh8, enabled on-column tag removal and purification based on Fh8 calcium-binding properties and ΔI-CM self-cleavage properties, and promoted the release of highly active protein with high yield and purity. Overall, our findings suggest that this Fh8-ΔI-CM system could be used as a novel solubility-inducing and purification fusion tag for protein production in E. coli.
Assuntos
Inteínas/genética , Proteínas dos Microfilamentos/química , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Sítios de Ligação , Escherichia coli/química , Escherichia coli/genética , Peptídeo Hidrolases/química , Potyvirus/química , Potyvirus/genética , Agregados Proteicos/genética , Solubilidade , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
The TNF-Related Apoptosis Inducing Ligand (TRAIL) cytokine triggers apoptosis specifically in cancer cells. Susceptibility of a given cell to TRAIL depends on the activity of regulatory proteins, one of the most important of which is BID. The aim of this study was to increase the cytotoxic potential of TRAIL against cancer cells. TRAIL was fused to the BH3 domain of BID. Hence, TRAIL acted not only as an anticancer agent, but also as a specific carrier for the BID fragment. Two fusion protein variants were obtained by genetic engineering, harboring two different linker sequences. The short linker allowed both parts of the fusion protein to fold into their native structures. The long linker influenced the structure of the fused proteins but nonetheless resulted in their highest cytotoxic activity. Optimal buffer formulation was determined for all the analyzed TRAIL variants. Fusing the BH3 domain of BID to TRAIL improved the cytotoxic potential of TRAIL. Further, these findings may be useful for the optimization of other anticancer drugs based on TRAIL, since the appropriate formulation would secure their native structures during prolonged storage.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Citotoxinas , Proteínas Recombinantes de Fusão , Ligante Indutor de Apoptose Relacionado a TNF , Células A549 , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/biossíntese , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/isolamento & purificação , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/farmacologia , Citotoxinas/biossíntese , Citotoxinas/química , Citotoxinas/isolamento & purificação , Citotoxinas/farmacologia , Células Hep G2 , Humanos , Domínios Proteicos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
TNF ligand superfamily member 10 (TRAIL) is a member of the tumor necrosis factor superfamily. The present study was performed in an effort to increase the expression of soluble (s)TRAIL by rebuilding the gene sequence of TRAIL. Three principles based on the codon bias of Escherichia coli were put forward to design the rebuild strategy. Relying on these three principles, a P7R mutation near the Nterminal region of sTRAIL, named TRAILMu, was designed. TRAILMu was subsequently cloned into the PTWIN1 plasmid and expressed in E. coli BL21 (DE3). Using a highlevel expression system and a threestep purification method, soluble TRAILMu protein reached ~90% of total cellular protein and purity was >95%, demonstrating success in overcoming inclusion body formation. The cytotoxic effect of TRAILMu was evaluated by sulforhodamine B assay in the MDMB231, A549, NCIH460 and L02 cell lines. The results demonstrated that TRAILMu exerted stronger antitumor effects on TRAILsensitive tumor cell lines, and was able to partially reverse the resistance of a TRAILresistant tumor cell line. In addition, TRAILMu exhibited no notable biological effects in a normal liver cell line. The novel TRAIL variant generated in the present study may be useful for the mass production of this important protein for therapeutic purposes.
Assuntos
Substituição de Aminoácidos , Arginina/genética , Códon , Mutação , Prolina/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. The aim of this study is to produce large quantities of highly pure and bioactive recombinant human TRAIL. Here, TRAIL was expressed in soluble form by pH-stat fed-batch cultivation and purified using a rapid and simple two-step chromatographic procedure. To improve the soluble yield, expression of TRAIL in Escherichia coli was induced with low IPTG concentration (0.1 mM) at low temperature (28 °C) supplemented with ZnSO4 (0.5 mM), using glycerol as carbon source. Under the optimized conditions, 4.14 ± 0.19 g/L of TRAIL in soluble form was achieved at 19 h without pure oxygen. To purify the recombinant TRAIL, we developed an efficient two-step chromatographic procedure including affinity chromatography and cation-exchange chromatography, especially improved the cation-exchange chromatography using a combination of pH and NaCl gradients strategy. Consequently, 4313.5 mg of target protein with high purity (98.1%) was obtained from 2.3 L of cell broth. Our results also showed that the purified TRAIL was with ordered secondary and tertiary structures, in homogeneous form and with strong cytotoxicity.
Assuntos
Ligante Indutor de Apoptose Relacionado a TNF , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificaçãoRESUMO
Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered an attractive anticancer agent due to its tumor cell-specific cytotoxicity. However, earlier studies with recombinant TRAIL revealed many shortcomings, including a short half-life, off-target toxicity and existence of TRAIL-resistant tumor cells. In this study, we developed a novel engineering strategy for recombinant soluble TRAIL by redesigning its structure with the adenovirus knobless fiber motif to form a stable homotrimer with improved antitumor activity. The result is a highly stable fiber-TRAIL fusion protein that could form homotrimers similar to natural TRAIL. The recombinant fusion TRAIL developed here displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. This construct will serve as a foundation for a new generation of recombinant proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL.
Assuntos
Adenoviridae/genética , Antineoplásicos/farmacologia , Engenharia Genética , Multimerização Proteica , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacocinética , Solubilidade , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacocinética , Distribuição Tecidual/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
VAS-TRAIL is a bifunctional fusion protein that combines anti-angiogenic activity with tumor-selective apoptotic activity for enhanced anti-tumor efficacy. VAS-TRAIL is expressed as inclusion body in Escherichia coli, but protein refolding is difficult to achieve and results in low yields of bioactive protein. In this study, we describe an efficient method for VAS-TRAIL refolding. The solubilization of aggregated VAS-TRAIL was achieved by a triple agent solution, which consists of an alkaline solution (pH 11.5) containing 0.4M l-arginine and 2M urea. The solubilized protein showed high purity and preserved secondary structure according to fluorescence properties. VAS-TRAIL refolding was performed through stepwise dialysis and resulted in more than 50% recovery of the soluble protein. The function of l-arginine was additive with alkaline pH, as shown by the significant improvement in refolding yield (≈30%) by l-arginine-containing solubilization solutions compared with alkaline solubilization solutions without l-arginine. The refolded VAS-TRAIL also showed ß-sheet structures and the propensity for oligomerization. Bioassays showed that the refolded fusion protein exhibited the expected activities, including its apoptotic activities toward tumor and endothelial cells, which proposed its promising therapeutic potential.
Assuntos
Calreticulina/química , Calreticulina/farmacologia , Corpos de Inclusão/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Redobramento de Proteína , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Arginina/química , Calreticulina/isolamento & purificação , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia em Gel , Cromatografia por Troca Iônica , Escherichia coli , Humanos , Fragmentos de Peptídeos/isolamento & purificação , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Solubilidade , Soluções , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ureia/químicaRESUMO
The structure of death receptor 4 (DR4) in complex with TNF-related apoptosis-inducing ligand (TRAIL) has been determined at 3â Å resolution and compared with those of previously determined DR5-TRAIL complexes. Consistent with the high sequence similarity between DR4 and DR5, the overall arrangement of the DR4-TRAIL complex does not differ substantially from that of the DR5-TRAIL complex. However, subtle differences are apparent. In addition, solution interaction studies were carried out that show differences in the thermodynamics of binding DR4 or DR5 with TRAIL.
Assuntos
Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/química , Sequência de Aminoácidos , Calorimetria , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , TermodinâmicaRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is capable of selectively inducing apoptosis of cancer cells, is a potential targeted drug for cancer therapy. The TRAIL protein induces apoptosis only in trimeric form. However, the recombinant soluble TRAIL (sTRAIL) trimer has low stability and a short half-life, which is a major obstacle for its advancement into clinical trials. Moreover, a percentage of engineered sTRAIL proteins are produced as dimers which may be toxic to normal human hepatocytes. In this study, we inserted three copies of the same subunit fragment of sTRAIL with a His tag into a polycistronic expression vector (pST39) to explore whether it would increase the proportion of trimers. We also constructed a heterozygous vector containing three subunit fragments of sTRAIL each with a different tag (His, HA, and Cmyc). Hybrid sTRAIL proteins (P-dTags) mainly as heterologous trimers were obtained by elution with a low concentration of imidazole based on different binding affinities of His with a nickel column. Functional analysis demonstrated that heterotrimeric forms of sTRAIL showed more stable activity compared to the P-3H at 4°C but not at 37°C without alteration in the native killing capacity. In addition, the heterologous trimers showed decreased toxicity to hepatocytes. These results suggest that the polycistronic expression system may be useful for expression of recombinant sTRAIL and improving its potential in cancer therapeutic applications.
Assuntos
Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Escherichia coli , Humanos , Plasmídeos/genética , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/genética , TemperaturaRESUMO
Apoptosis and necroptosis are dependent on the formation/activation of distinct multi-protein complexes; these include the Death-Inducing Signalling Complex (DISC), apoptosome, piddosome, necrosome and ripoptosome. Despite intense research, the mechanisms that regulate assembly/function of several of these cell death signalling platforms remain to be elucidated. It is now increasingly evident that the composition and stoichiometry of components within these key signalling platforms not only determines the final signalling outcome but also the mode of cell death. Characterising these complexes can therefore provide new insights into how cell death is regulated and also how these cell death signalling platforms could potentially be targeted in the context of disease. Large multi-protein complexes can initially be separated according to their size by gel filtration or sucrose density gradient centrifugation followed by subsequent affinity-purification or immunoprecipitation. The advantage of combining these techniques is that you can assess the assembly of individual components into a complex and then assess the size and stoichiometric composition of the native functional signalling complex within a particular cell type. This, alongside reconstitution of a complex from its individual core components can therefore provide new insight into the mechanisms that regulate assembly/function of key multi-protein signalling complexes. Here, we describe the successful application of a range of methodologies that can be used to characterise the assembly of large multi-protein complexes such as the apoptosome, DISC and ripoptosome. Together with their subsequent purification and/or reconstitution, these approaches can provide novel insights into how cell death signalling platforms are regulated in both normal cell physiology and disease.
Assuntos
Apoptose/genética , Apoptossomas/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/isolamento & purificação , Linfócitos/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Apoptossomas/metabolismo , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Regulação da Expressão Gênica , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Multimerização Proteica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in Escherichia coli as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.
Assuntos
Colágeno Tipo XVIII/metabolismo , Dobramento de Proteína , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Cromatografia em Gel , Colágeno Tipo XVIII/genética , Colágeno Tipo XVIII/isolamento & purificação , Colágeno Tipo XVIII/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Escherichia coli/genética , Expressão Gênica , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
AIM: To construct the expression vector pET-28α-Trail(114-281); and find the optimal conditions for target gene expression, host bacteria lysis, and protein purification, and to detect the apoptosis function of the recombinant protein. METHODS: The functional domain of Trail(114-281); was amplified by PCR and cloned into the expression vector pET-28α(+). After confirmed by DNA sequencing, the Trail(114-281); was expressed in E.coli BL21 under the condition of different A(600);, IPTG concentration and temperature. Host bacteria were lysed using three different ways, including ultrasonication, osmotic shock and IP lysis, and the target protein was purified using Ni-NTA affinity chromatography or cutting-gel purification. The advantages and shortcomings of these methods were compared to find the most efficient ways for expression and purification of the recombinant protein. The immunocompetence of Trail protein from cutting-gel purification was analyzed by Western blotting, A549 cell apoptosis induced by purified protein from Ni-NTA chromatography was detected by flow cytometry. RESULTS: The 516 bp Trail(114-281); gene was cloned, and expressed in E.coli BL21. When A(600);=0.6, recombinant host bacteria were induced by 1.0 mmol/L IPTG at 37 DegreesCelsius for 4 h, which was the optimal condition for the expression of inclusion body, and the soluble protein was expressed stably on the condition of 25 DegreesCelsius, A(600);=1.0, IPTG1.0 mmol/L. Ultrasonication could get maximal protein compared to the other methods. The two purification ways both could purify taget protein successfully. Western blot analysis showed that the protein purified by cutting-gel has a good immunologic activity. Protein from Ni-NTA affinity chromatography caused cell apoptosis. CONCLUSION: The expression vector pET-28α-Trail(114-281); can be constructed and expressed in E.coli BL21 successfully. Temperature is a more important effect factor of Trail(114-281); expression in host bacteria compared with other factors. Cutting-gel protein has immunogenicity, and Ni-NTA protein could keep its function. These results provide a basis for the further functional research and application of Trail.
Assuntos
Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Solubilidade , Ligante Indutor de Apoptose Relacionado a TNF/químicaRESUMO
AIMS AND BACKGROUND: As a powerful technique allowing analysis of large numbers of cells, fluorescence-activated cell sorting (FACS) is used more and more widely. For FACS analysis, adherent cells are usually detached by trypsinization, followed by centrifugation and resuspension. However, trypsinization can cut off some receptors from the cell surface like fine scissors, which will affect the accuracy of FACS results. Though non-enzymatic methods such as citric saline buffer have been used to determine cell surface receptors, how much of the receptors is cut off by trypsinization has been rarely studied. This work aimed to investigate whether different methods of detaching adherent cells could affect the detection of cell surface receptors. METHODS: Human hepatocellular carcinoma cell lines (HepG2, Huh7 and Hep3B) were detached enzymatically with trypsin-EDTA solution or non-enzymatically with citric saline buffer, and then the receptors of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were detected by FACS analysis. Cell viability, cell cycle and apoptosis (sub-G1 fraction detected by FACS) of the trypsin-EDTA group and citric saline buffer group were also studied. RESULTS: Different methods of detaching adherent cells could significantly affect the detection of TRAIL receptors. Compared to the conventional trypsin-EDTA group, the non-enzymatic group showed a 3.42-fold increase in the mean fluorescence intensity index of DcR HepG2 and a 1.25-fold increase in DR Huh 7 (P <0.05). However, the viability, cell cycle and apoptosis of these cells were not affected. CONCLUSIONS: Citric saline buffer might be recommended as the first choice to detach adherent cells for FACS analysis of cell surface receptors.
Assuntos
Carcinoma Hepatocelular/química , Citometria de Fluxo/métodos , Neoplasias Hepáticas/química , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ácido Aconítico , Apoptose , Adesão Celular , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Ácido Edético , Humanos , Cloreto de Sódio , Ligante Indutor de Apoptose Relacionado a TNF/análise , TripsinaRESUMO
In this study, the complementary DNA (cDNA) of Japanese pufferfish Takifugu rubripes tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) was cloned by reverse-transcription PCR. The open reading frame of the TRAIL consisted of 870 bases. The deduced amino-acid sequence of the TRAIL showed a high homology with the sequences of other teleosts. Recombinant soluble TRAIL was fused with a small ubiquitin-related modifier gene to enhance the soluble expression level in Escherichia coli BL21 (DE3). In vitro, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assay indicated that the purified soluble TRAIL was able to induce apoptosis of Jurkat and HeLa cells in a dose-dependent manner.
Assuntos
Proteínas de Peixes/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Takifugu/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Peixes/isolamento & purificação , Proteínas de Peixes/metabolismo , Componentes do Gene , Células HeLa , Humanos , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Takifugu/metabolismoRESUMO
BACKGROUND: The search for strategies to target ion channels for therapeutic applications has become of increasing interest. Especially, the potassium channel K(V)10.1 (Ether-á-go-go) is attractive as target since this surface protein is virtually not detected in normal tissue outside the central nervous system, but is expressed in approximately 70% of tumors from different origins. METHODS: We designed a single-chain antibody against an extracellular region of K(V)10.1 (scFv62) and fused it to the human soluble TRAIL. The K(V)10.1-specific scFv62 antibody -TRAIL fusion protein was expressed in CHO-K1 cells, purified by chromatography and tested for biological activity. RESULTS: Prostate cancer cells, either positive or negative for K(V)10.1 were treated with the purified construct. After sensitization with cytotoxic drugs, scFv62-TRAIL induced apoptosis only in K(V)10.1-positive cancer cells, but not in non-tumor cells, nor in tumor cells lacking K(V)10.1 expression. In co-cultures with K(V)10.1-positive cancer cells the fusion protein also induced apoptosis in bystander K(V)10.1-negative cancer cells, while normal prostate epithelial cells were not affected when present as bystander. CONCLUSIONS: K(V)10.1 represents a novel therapeutic target for cancer. We could design a strategy that selectively kills tumor cells based on a K(V)10.1-specific antibody.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Antineoplásicos/isolamento & purificação , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/genética , Efeito Espectador , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Canais de Potássio Éter-A-Go-Go/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificaçãoRESUMO
Members of the tumor necrosis factor (TNF) superfamily influence a variety of immunological functions, including cellular activation, proliferation, and death, upon interaction with a corresponding superfamily of receptors. Whereas interest in the apoptosis-inducing molecules TNF and Fas ligand has peaked because of their participation in events such as autoimmune disorders, activation-induced cell death, immune privilege, and tumor evasion from the immune system, another death-inducing family member, TNF-related apoptosis-inducing ligand (TRAIL), or Apo-2 ligand, has generated excitement because of its unique ability to induce apoptosis in a wide range of transformed cell lines but not in normal tissues. TRAIL is well tolerated when given to healthy animals, and no observable histological or functional changes have been observed in any of the tissues or organs examined. Moreover, multiple injections of soluble TRAIL into mice beginning the day after tumor implantation can significantly suppress the growth of the tumors, with many animals becoming tumor-free. One potential drawback to these findings is that large amounts of soluble TRAIL may be required to inhibit tumor formation, possibly because of the pharmacokinetic profile of soluble TRAIL that indicates that, after intravenous injection, the majority of the protein is rapidly cleared. Increasing the in vivo half-life of recombinant soluble TRAIL or developing an alternative means of delivery may increase the relative tumoricidal activity of TRAIL such that larger, more established tumors could be eradicated as efficiently as smaller tumors. The information presented here describes the production of an adenoviral vector engineered to carry the complementary DNA (cDNA) for murine TRAIL (hTRAIL).
Assuntos
Apoptose , Terapia Genética/métodos , Neoplasias/patologia , Neoplasias/terapia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Adenoviridae/genética , Animais , Extratos Celulares , Linhagem Celular Tumoral , Proliferação de Células , Exocitose , Humanos , Camundongos , Neoplasias/genética , Fosfatidilserinas/metabolismo , Reprodutibilidade dos Testes , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Transfecção , Transgenes , Vírion/isolamento & purificaçãoRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the TNF superfamily. In this paper, we report the expression, purification, and preparation of a recombinant form of the extracelluar domain of the TRAIL (sTRAIL) without posttranslational modifications, which may selectively induce apoptosis of tumor cells in vitro. To obtain recombinant nonfusion sTRAIL protein, the encoding region for sTRAIL was cloned between KpnI and BamHI in pET32a. The Trx (thioredoxin)/sTRAIL fusion proteins were expressed in the form of inclusion bodies in Escherichia coli host strain BL21 (DE3). The expression level was more than 35% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2 M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >98% were obtained. Trx/sTRAIL L proteins were digested by enterokinase to both Trx and sTRAIL fragments, which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rsTRAIL (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the recombinant sTRAIL may have cancer therapeutic applications.
Assuntos
Escherichia coli/genética , Regulação da Expressão Gênica , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/química , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Corpos de Inclusão , Plasmídeos/genética , Dobramento de Proteína , RNA/sangue , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/biossínteseRESUMO
The human TRAIL gene (encoding residues 114-281) was synthesized by PCR and cloned into plasmid pET-32a. High level expression (1.5 g l(-1)) of thioredoxin/TRAIL fusion was achieved in Escherichia coli strain BL21(DE3), mainly as inclusion bodies. Refolded fusion thioredoxin/TRAIL was cleaved by enteropeptidase and TRAIL was separated from thioredoxin on Ni-NTA agarose. High yield (400 mg l(-1)) of TRAIL without N-terminal methionine and His tag was obtained. Sedimentation coefficient demonstrated that 98% of TRAIL formed trimers. TRAIL formed crystals of space group P3 (1) with unit-cell dimensions a = b = 72.5 A, c = 141.5 A. Apoptosis induced in HeLa cells by purified TRAIL was 5-fold enhanced by emetine.
Assuntos
Enteropeptidase/metabolismo , Corpos de Inclusão/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Tiorredoxinas/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Cristalografia , Escherichia coli/genética , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Tiorredoxinas/genéticaRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the TNF superfamily. Here, a recombinant form of the extracellular domain of the TRAIL (sTRAIL) was expressed in Escherichia coli BL21(DE3) under the control of a T7 promoter. The resulting insoluble bodies were separated from cellular debris by centrifugation and solubilized with 8 M urea. A rapid and simple on-column refolding procedure was developed. It was applied and then the refolded sTRAIL was purified by anion-exchange chromatography. The purified final product was >98% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 19.2 kDa, which equalled the theoretically expected mass. N-terminal sequencing of refolding sTRAIL showed the sequence which corresponded to the designed protein. The renatured protein displayed its immunoreactivity with the antibodies to TRAIL protein by Western blotting. The purified sTRAIL had a strong cytotoxic activity against human cervical cancer HeLa cells with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with beta-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.