Gotowa bibliografia na temat „Recombinant truncated thioredoxin”

A thioredoxin (BbTrx) was identified from the entomopathogenic fungus Beauveria bassiana . The cloned nucleotide sequence consisted of a 423-bp open reading frame encoding a 141-amino-acid thioredoxin, a 1011-bp 5′ region, and a 419-bp 3′ region. The deduced protein sequence of BbTrx, including a common 95-amino-acid conserved domain and a unique 46-amino-acid carboxy terminal region, was similar (≤38% identity) to that of other thioredoxins and phylogenetically closest to that from Neurospora crassa . In insulin solution containing dithiothreitol at 25 °C, recombinant BbTrx or a truncated form lacking the carboxy terminal region (BbTrxD) exhibited disulfide reduction activity. BbTrxD was more active after pre-incubation at 40–75 °C, and cells expressing BbTrxD showed significantly higher tolerance to thermal stress (51 °C). The BbTrx expression in B. bassiana was greatly elevated when stressed at 40 °C. The results indicate that the new thioredoxin is a potential target for improving the thermotolerance of B. bassiana formulations.

Background: Insulin is a peptide hormone used for regulating blood glucose levels. Human insulin market is projected to grow at a rate of 12.5% annually. To meet the needs of patients, a cost effective insulin manufacturing strategy has to be developed. This can be achieved by selecting a competent host, ideal fusion tag and streamlined downstream process. Objective: In this article, we have demonstrated that selecting a right fusion partner for expression of toxic proteins like insulin, plays a major role in increasing the recombinant protein yield. Methods: In this article, we have focused on identifying a peptide tag fusion partner for expressing proinsulin by truncating thioredoxin tag. Truncations were carried out from both Amino and Carboxy terminus of the protein and efficiency of truncated sequences was evaluated by expressing it with proinsulin gene. FCTRX (1-15) sequence fused to proinsulin was processed further to establish downstream protocol for purification. Results: Thioredoxin tag was truncated appropriately by considering the fusion tag: protein ratio. A couple of sequences ranging 10 – 15 amino acids were identified based on its in silico properties. Of these FCTRX (1-15) showed increased expression and stability of fusion protein. 156 mg of purified insulin was generated from 1g of inclusion body after enzymatic conversion and chromatographic steps. Conclusion: As a result of the current study, it was concluded that FCTRX (1-15) peptide has advantageous attributes to be considered as an ideal fusion tag for expression of proinsulin. This can be further explored by expressing it with other proteins.

Abstract Human thioredoxin (Trx) is the major 12-kd cellular disulfide-reductase that on secretion acts as a cocytokine with several interleukins. Truncated Trx with the 80 N-terminal residues (Trx80), also present in plasma, was by itself a mitogenic cytokine for human peripheral blood mononuclear cells (PBMC). This study investigated which cells in PBMC are targets of recombinant Trx80. Purified human CD14+ monocytes, but not B or T cells, in a synthetic medium were activated to differentiation by Trx80 as measured by flow cytometry of surface antigens because exposure to 100 nM Trx80 increased expression of CD14, CD40, CD54, and CD86. Proliferation of the monocytes was increased in a dose-dependent manner by Trx80 in concentrations ranging from 10 nM to 1 μM. Trx or interleukin (IL) 2 did not induce proliferation or expression of surface antigens on monocytes. Trx80 alone induced secretion of IL-12 from CD40+ monocytes in the PBMC cultures and this effect was enhanced by IL-2. Trx80 and IL-2 together were strongly synergistic to induce secretion of interferon-γ in PBMC cultures. The results showed that Trx80 is a potent cytokine for normal human monocytes and directs the immune system in favor of a Th1 response via IL-12 production.

Piperlongumine, a natural alkaloid substance extracted from the fruit of the long pepper (Piper longum Linn.), is known to inhibit the cytosolic thioredoxin reductase (TXNRD1 or TrxR1) and selectively kill cancer cells. However, the details and mechanism of the inhibition by piperlongumine against TXNRD1 remain unclear. In this study, based on the classical DTNB reducing assay, irreversible inhibition of recombinant TXNRD1 by piperlongumine was found and showed an apparent kinact value of 0.206 × 10−3 µM−1 min−1. Meanwhile, compared with the wild-type TXNRD1 (-GCUG), the UGA-truncated form (-GC) of TXNRD1 was resistant to piperlongumine, suggesting the preferential target of piperlongumine is the selenol (-SeH) at the C-terminal redox motif of the enzyme. Interestingly, the high concentration of piperlongumine-inhibited TXNRD1 showed that its Sec-dependent activity is decayed but its intrinsic NADPH oxidase activity is retained. Furthermore, piperlongumine did not induce ferroptosis in HCT116 cells at 10 µM, whereas significantly promoted erastin-induced lipid oxidation, which could be alleviated by supplying glutathione (GSH) or N-acetyl L-cysteine (NAC). However, restricting GSH synthesis by inhibiting glutaminase (GLS) using the small molecule inhibitor CB-839 only slightly enhanced erastin-induced cell death. Taken together, this study elucidates the molecular mechanism of the antitumor capacity of piperlongumine by targeting TXNRD1 and reveals the potential possibility of inhibiting TXNRD1 to strengthen cancer cells’ ferroptosis.

Over-expression of genetically encoded thioredoxin reductase 1 (TrxR1) TrxR1 can be toxic to cells due to the formation of a truncated version of the enzyme. We developed a new mammalian cell-based model to investigate TrxR1 activity. Fusion of the HIV-derived cell penetrating peptide (TAT) enabled efficient cellular uptake of purified TrxR1 containing 21 genetically encoded amino acids, including selenocysteine. The TAT peptide did not significantly alter the catalytic activity of TrxR1 in vitro. We monitored TrxR1-dependent redox activity in human cells using a TrxR1-specific red fluorescent live-cell reporter. Using programmed selenocysteine incorporation in Escherichia coli, our approach allowed efficient production of active recombinant human selenoprotein TrxR1 for delivery to the homologous context of the mammalian cell. The delivered TAT-TrxR1 showed robust activity in live cells and provided a novel platform to study TrxR1 biology in human cells.

The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.

The experiments described in this thesis involve the expression and characterisation of recombinant truncated thioredoxin (tTrx) and the potential involvement that thioredoxin (Trx) has in the cellular responses to oxidative stress. Truncated Trx (80 amino acids) was expressed from a plasmid containing the ORF for tTrx that had been introduced into E.coli BL-21(DE3) cells. The protein was initially extracted using a combination of high concentrations of urea, high pH levels, and multiple sonification steps to remove the tTrx from inclusion bodies formed during expression. This procedure produced a stable solution of tTrx. Purification of tTrx from this protein solution required anion exchange chromatography followed by gel permeation in a HPLC system to obtain fully purified, recombinant tTrx which allowed further characterisation studies to be undertaken. An initial investigation into tTrx was performed to determine some basic physical, biochemical and functional aspects of this hitherto relatively undefined protein. Analysis by sedimentation equilibrium indicated that freshly prepared tTrx forms a single species with a molecular weight of 18.8kDa. This value indicates that recombinant tTrx naturally forms a dimer in solution that was shown to be non-covalent in nature and stable in solution. The capacity of tTrx to reduce protein disulphide bonds was determined using the insulin reduction assay. Results show that tTrx lacks this particular redox ability. The rate of oxidisation at 4 degrees C was analysed using free thiol determination, sedimentation equilibrium and SDS-PAGE patterning. Results indicated a steady rise in the degree of oxidation of tTrx over an eight day period. After six days the oxidated protein consistently displayed the presence of intramolecular disulphide bonds. Covalently-linked disulphide dimers and higher molecular weight oligomers were detectable after eight days oxidation. An investigation of the reducing capacity of the basic Trx system determined that fully oxidised tTrx was unable to act alone as a substrate for thioredoxin reductase (TR). However, when reduced Trx was added to the system, it appeared capable of acting as an electron donor to the oxidised tTrx in order to reduce disulphide groups. Recombinant tTrx was successfully radiolabelled with Trans 35S-methionine/cysteine for use in cell association studies. No evidence was found to indicate the presence of a receptor for tTrx on either MCF-7 or U-937 cells. Findings suggest that a low level of non-specific binding of tTrx to these cell lines rather than a classical ligand-binding mechanism occurs thus suggesting the absence of a cell surface receptor for tTrx. The role that Trx may play in the cellular responses to oxidative stress was also investigated. The chemical oxidants hydrogen peroxide (H2O2) and diamide were used to establish an in vitro model of oxidative stress for the choriocarcinoma cytotrophoblast cell line JEG-3. Cellular function was assessed in terms of membrane integrity, metabolic activity and the ability to synthesis new DNA following exposure to these oxidants. Results indicated that both agents were capable of causing cells to undergo oxidative stress without inducing immediate apoptosis or necrosis. Initially, JEG-3 cells exposed to 38μM or 75μM H2O2 or 100μM diamide were shown to display altered cell metabolism and DNA synthesis without loss to cell viability or membrane integrity. Cells were also shown to be capable of some short-term recovery but later lapsed into a more stressed state. Expression levels of Trx were studied to determine whether this type of chemical stress caused a change in intercellular protein levels. Both cELISA and western blotting results indicated that only cells exposed to 100μM diamide displayed any significant increase in Trx protein levels after 6 or 8hrs exposure to the oxidant. Further studies over a longer time-frame were also performed. These found that when JEG-3 cells were exposed to 18μM H2O2 or 200μM diamide over 12-48hrs, a positive correlation between increasing endogenous Trx protein levels and a decline in cell proliferation was observed. Cytotrophoblast cells, which are responsible for implantation and placentation, are susceptible to oxidative stress in vivo and their anti-oxidant capacity is fundamental to the establishment of pregnancy. The findings obtained during these studies suggest that Trx plays a role in this process.
Thesis (Masters)
Master of Philosophy (MPhil)
School of Health Sciences
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