Cloning, Functional Characterization and Response to Cadmium Stress of the Thioredoxin-like Protein 1 Gene from Phascolosoma esculenta.

Cadmium (Cd) is a heavy metal toxicant and is widely distributed in aquatic environments. It can cause excessive production of reactive oxygen species (ROS) in the organism, which in turn leads to a series of oxidative damages. Thioredoxin (Trx), a highly conserved disulfide reductase, plays an important role in maintaining the intracellular redox homeostasis in eukaryotes and prokaryotes. Phascolosoma esculenta is an edible marine worm, an invertebrate that is extensively found on the mudflats of coastal China. To explore the molecular response of Trx in mudflat organisms under Cd stress, we identified a new Trx isoform (Trx-like protein 1 gene) from P. esculenta for the first time, designated as PeTrxl. Molecular and structural characterization, as well as multiple sequence and phylogenetic tree analysis, demonstrated that PeTrxl belongs to the Trx superfamily. PeTrxl transcripts were found to be ubiquitous in all tissues, and the highest expression level occurred in the coelomic fluid. Exposure to three sublethal concentrations of Cd resulted in the upregulation and then downregulation of PeTrxl expression levels over time in coelomic fluid of P. esculenta. The significant elevation of PeTrxl expression after 12 and 24 h of Cd exposure at 6 and 96 mg/L, respectively, might reflect its important role in the resistance to Cd stress. Recombinant PeTrxl (rPeTrxl) showed prominent dose-dependent insulin-reducing and ABTS free radical-scavenging abilities. After exposure to 96 mg/L Cd for 24 h, the ROS level increased significantly in the coelomic fluid, suggesting that Cd induced oxidative stress in P. esculenta. Furthermore, the injection of rPeTrxl during Cd exposure significantly reduced the ROS in the coelomic fluid. Our data suggest that PeTrxl has significant antioxidant capacity and can protect P. esculenta from Cd-induced oxidative stress.

AtTrxh3, a Thioredoxin, Is Identified as an Abscisic AcidBinding Protein in Arabidopsis thaliana.

Abscisic acid (ABA, 1) is a plant hormone that regulates various plant physiological processes such as seed developing and stress responses. The ABA signaling system has been elucidated; binding of ABA with PYL proteins triggers ABA signaling. We have previously reported a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, a protein cross-linker, and a bioactive small molecule with an azido group (azido probe). This method was used to identify the unknown ABA binding protein of Arabidopsis thaliana. As a result, AtTrxh3, a thioredoxin, was isolated as an ABA binding protein. Our developed method can be applied to the identification of binding proteins of bioactive compounds.

Expression of rice ß-exoglucanase II (OsExoII) in Escherichia coli, purification, and characterization.

Enzymes involved in ß-glucan breakdown in plants include endoglucanases, exoglucanases and ß-glucosidases. Glycoside hydrolase family 3 (GH3) exoglucanases from barley and maize and a few plant GH3 ß-glucosidases have been characterized, but none from rice. A few of these enzymes have been expressed in recombinant yeast and plant systems, but bacterial expression of plant GH3 enzymes has not been successful. We expressed the rice GH3 exoglucanase OsExo2 in Escherichia coli as a thioredoxin fusion protein, while other active plant GH3 enzymes could not be produced in this system. The protein was purified over 2000-fold in three chromatographic steps. The enzyme hydrolyzed ß-1,3- and ß-1,4-linked oligosaccharides and polysaccharides, consistent with a role in cell wall remodeling. Of the oligosaccharides tested, it had highest catalytic efficiency toward laminaritriose, (apparent kcat/Km = 37.7 mM-1s-1). Among polysaccharides, OsExoII hydrolyzed barley mixed ß-glucan and laminarin with similar efficiencies (apparent kcat/Km = 3.7 and 3.4 mL mg-1 s-1, respectively), but achieved its highest apparent kcat with lichenan (2.9 s-1). OsExoII was found to be stimulated by ethylene glycol, which increased the apparent kcat and decreased the Km and was transglycosylated. These results imply that E. coli expression may be successful for certain plant GH3 enzymes and OsExoII may be a useful enzyme for application to glycoside production.

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding.

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionarily conserved, but span at least a ∼100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.

Identification of thioredoxin domain-containing protein 17 from big-belly seahorse Hippocampus abdominalis: Molecular insights, immune responses, and functional characterization.

Thioredoxin domain-containing protein 17 (TXNDC17) is a small protein (∼14 kDa) involved in maintaining cellular redox homeostasis via a thiol-disulfide reductase activity. In this study, TXNDC17 was identified and characterized from Hippocampus abdominalis. The open reading frame (ORF) consisted of 369 bp and 123 amino acids. Similar to the other thioredoxins, TXNDC17 contained a conserved WCXXC functional motif. The highest spatial mRNA expressions of HaTXNDC17 were observed in the muscle, brain, and intestine. Interestingly, the mRNA expression of HaTXNDC17 in blood showed significant upregulation at 48 h against all the pathogen associated molecular patterns (PAMPs) and bacteria. Further, HaTXNDC17 transcripts in the trunk kidney were significantly upregulated at 24-48 h by bacterial endotoxin lipopolysaccharides (LPS), viral mimic polyinosinic: polycytidylic acid (poly I:C), and gram-negative bacteria (Edwardsiella tarda). The DPPH assay showed that the radical scavenging activity varies in a concentration-dependent manner. The insulin reduction assay demonstrated a significant logarithmic relationship with the concentration of rHaTXNDC17. Moreover, FHM cells treated with recombinant HaTXNDC17 significantly enhanced cellular viability under oxidative stress. Together, these results show that HaTXNDC17 function is important for maintaining cellular redox homeostasis and that it is also involved in the immune mechanism in seahorses.

Cloning, expression, purification, and kinetic characterization of mitochondrial thioredoxin (TsTrx2), cytosolic thioredoxin (TsTrx1), and glutaredoxin (TsGrx1) from Taenia solium.

We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.

Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus.

In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction. The protein purification was performed by nickel affinity chromatography and DEAE anion exchange chromatography. The purified product has a purity of 95% with a yield of 47.3 mg/L of culture. The specific activity of the fusion protein reaches to 2.0 × 107 IU/mg as determined in the CEF/VSV titration system. After excision of the Trx tag by enterokinase, the remaining solo protein was confirmed as rChIFN-α protein by SDS-PAGE, N-terminal sequencing and mass spectrometry. The effects of this Trx-rChIFN-α fusion protein against H9N2 influenza virus infection were also evaluated in ovo. The results showed that the Trx-rChIFN-α protein could significantly reduce the hemagglutination titer of H9N2 virus, and the H9N2 viruses HA gene copy numbers. These findings will enable us to produce large amount and bio-active rChIFN-α protein for future applications.

Role of the glutaredoxin domain and FAD in the stabilization of thioredoxin glutathione reductase.

Thioredoxin glutathione reductase (TGRsec) is a multi-domain flavoprotein that plays a principal role in redox homeostasis maintenance. We have previously demonstrated the role of selenocysteine in maintaining TGRsec structure-function, but the role of the glutaredoxin (Grx) domain and FAD is still unclear. In the present study, the urea-induced unfolding of recombinant Fasciola gigantica TGRsec (FgTGRsec) and its N-terminal truncated variant (ΔNTD-FgTGRsec) were examined to understand the role of the Grx domain and FAD in the stabilization of FgTGRsec and ΔNTD-FgTGRsec. Our results showed that both proteins underwent unfolding in a three state manner. First, the protein undergoes a conformational transition rendering a near-native state with no FAD bound, and then full unfolding of the apo-dimer occurs without dissociation. The Grx domain stabilized the global FgTGRsec structure and positively regulated FgTGRsec activity, and alteration in the FAD microenvironment was directly proportional to the loss of thioredoxin reductase (TrxR) and glutathione reductase activities. Based on these results, we concluded that the Grx domain stabilizes the full-length FgTGRsec protein for efficient catalysis. Thus, we suggest that in platyhelminth parasites, during evolution, the Grx domain merged with the TrxR domain to confer higher catalytic activity and provide additional structural stability to the full-length TGR.

Remote ischemic preconditioning protects human neural stem cells from oxidative stress.

In previous clinical trials, we showed that remote ischemic preconditioning (rIPC) reduced myocardial damage in children undergoing treatment for congenital heart defects and postoperative renal failure in patients undergoing abdominal aortic aneurysm surgery. In rabbit experiments, pre-treatment with plasma and plasma dialysate (obtained using 15-kDa cut-off dialysis membrane) from donor rabbits subjected to rIPC similarly protected against cardiac infarction. However, the protective substances containing in rIPC plasma have been unknown. In the present study, we showed that rIPC plasma exerted anti-apoptotic and anti-oxidative effects on human neural stem cells under oxygen glucose deprivation (OGD) that mimics brain ischemia. Additionally, we applied the sample to the liquid chromatography integrated with mass spectrometry to identify candidate key molecules in the rIPC plasma and determine its role in protecting neural stem cells from OGD-induced cell death. Thioredoxin increased significantly after rIPC compared to pre-IPC. Pretreatment with thioredoxin, the antioxidant protein, markedly protected human neural stem cells from OGD-induced cell death. The effect of thioredoxin on brain ischemia in animals should be further evaluated. However, the present study first evaluated the effect of rIPC in the ischemic cellular model.

Secretory production of designed multipeptides displayed on a thermostable bacterial thioredoxin scaffold in Pichia pastoris.

Internal grafting of designed peptides to scaffold proteins is a valuable strategy for a variety of applications including recombinant peptide antigen construction. A peptide epitope from human papillomavirus (HPV) minor capsid protein L2 displayed on thioredoxin (Trx) has been validated preclinically as a broadly protective and low-cost alternative HPV vaccine. Focusing on thioredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus (PfTrx) as a scaffold, we have constructed a modified Pichia pastoris expression vector and used a PfTrx fusion derivative containing three tandemly repeated copies of a 19 amino acids peptide epitope from HPV-L2 for expression optimization and biochemical-immunological characterization of the Pichia-produced PfTrx-L2 antigen. We show that PfTrx-L2 is produced at high levels (up to 100 mg from a 100 ml starting culture using a multi-cycle induction protocol) and secreted into the culture medium as a highly enriched (>70% pure), non-glycosylated polypeptide that can be purified to homogeneity in a single step. Oxidation and aggregation state, thermal stability and immunogenicity of the endotoxin-free PfTrx-L2 antigen produced in P. pastoris were tested and found to be identical to those of the same antigen produced in Escherichia coli. Secretory production of endotoxin-free PfTrx-peptides in P. pastoris represents a cost- and time-effective alternative to E. coli production. Specifically designed for peptide antigens, the PfTrx-expression vector and conditions described herein are easily transferable to a variety of applications centred on the use of structurally constrained bioactive peptides as immune as well as target-specific binder reagents.

Efficient expression of recombinant human heavy chain ferritin (FTH1) with modified peptides.

Human heavy chain ferritin (FTH1) can self-assemble into a diameter of 12-nm spherical cage with an interior cavity of 8 nm in diameter. FTH1 has great potential as a nanocage in molecular imaging and drug delivery. Different peptides have been fused with FTH1 for targeting delivery; however, the expression of FTH1 modified with peptides in soluble form is not equivalent to natural FTH1. As shown in recent study, a novel scaffold protein --thioredoxin from the archaebacterium Pyrococcus furiosus (PfTrx)--exhibits a superior solubilization capacity and thermal stability [19]. Here we report a new construct (FTH1-PfTrx-His) that can be easily expressed and purified in Escherichia coli. Of note, different peptides inserted into FTH1-PfTrx-His did not influence the expression of proteins. Finally, the doxorubicin packaging ability of FTH1-PfTrx-His is comparable to natural FTH1. Our results showed that FTH1-PfTrx-His had a potential role as a novel peptide-modified ferritin carrier for drugs or imaging probes.

Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli.

Matrix metalloproteinases (MMPs) are crucial proteases in maintaining the health and integrity of many tissues, however their dysregulation often facilitates disease progression. In disease states these remodeling and repair functions support, for example, metastasis of cancer by both loosening the matrix around tumors to enable cellular invasion and by affecting proliferation and apoptosis, and they promote degradation of biological restorations by weakening the substrate to which the restoration is attached. As such, MMPs are important therapeutic targets. MMP-8 participates in cancer, arthritis, asthma and failure of dental fillings. MMP-8 differs from other MMPs in that it has an insertion that enlarges its active site. To elucidate the unique features of MMP-8 and develop selective inhibitors to this therapeutic target, a stable and active form of the enzyme is needed. MMP-8 has been difficult to express at high yield in a soluble, active form. Typically recombinant MMPs accumulate in inclusion bodies and complex methods are applied to refold and purify protein in acceptable yield. Presented here is a streamlined approach to produce in Escherichia coli a soluble, active, stable MMP-8 fusion protein in high yield. This fusion shows much greater retention of activity when stored refrigerated without glycerol. A variant of this construct that contains the metal binding claMP Tag was also examined to demonstrate the ability to use this tag with a metalloprotein. SDS-PAGE, densitometry, mass spectrometry, circular dichroism spectroscopy and an activity assay were used to analyze the chemical integrity and function of the enzyme.

A thioredoxin-like/ß-propeller protein maintains the efficiency of light harvesting in Arabidopsis.

The light-harvesting complexes of plants have evolved the ability to switch between efficient light harvesting and quenching forms to optimize photosynthesis in response to the environment. Several distinct mechanisms, collectively termed "nonphotochemical quenching" (NPQ), provide flexibility in this response. Here we report the isolation and characterization of a mutant, suppressor of quenching 1 (soq1), that has high NPQ even in the absence of photosystem II subunit S (PsbS), a protein that is necessary for the rapidly reversible component of NPQ. The formation of NPQ in soq1 was light intensity-dependent, and it exhibited slow relaxation kinetics and other characteristics that distinguish it from known NPQ components. Treatment with chemical inhibitors or an uncoupler, as well as crosses to mutants known to affect other NPQ components, showed that the NPQ in soq1 does not require a transthylakoid pH gradient, zeaxanthin formation, or the phosphorylation of light-harvesting complexes, and it appears to be unrelated to the photosystem II damage-and-repair cycle. Measurements of pigments and chlorophyll fluorescence lifetimes indicated that the additional NPQ in soq1 is the result of a decrease in chlorophyll excited-state lifetime and not pigment bleaching. The SOQ1 gene was isolated by map-based cloning, and it encodes a previously uncharacterized thylakoid membrane protein with thioredoxin-like and ß-propeller domains located in the lumen and a haloacid-dehalogenase domain exposed to the chloroplast stroma. We propose that the role of SOQ1 is to prevent formation of a slowly reversible form of antenna quenching, thereby maintaining the efficiency of light harvesting.

Expression of bioactive soluble human stem cell factor (SCF) from recombinant Escherichia coli by coproduction of thioredoxin and efficient purification using arginine in affinity chromatography.

Stem cell factor (SCF) known as the c-kit ligand is a two disulfide bridge-containing cytokine in the regulation of the development and function of hematopoietic cell lineages and other cells such as mast cells, germ cells, and melanocytes. The secreted soluble form of SCF exists as noncovalently associated homodimer and exerts its activity by signaling through the c-Kit receptor. In this report, we present the high level expression of a soluble recombinant human SCF (rhSCF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hSCF cDNA was cloned into pET3b vector, and transformed into E. coli BL21(DE3) harboring a bacterial thioredoxin coexpression vector. The recombinant protein was purified via an affinity chromatography processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Although almost none of the soluble fusion protein bound to the resin in standard protocol using 0.1M sodium phosphate buffer (pH 7.2), the use of binding buffer containing 0.5M l-arginine for protein stabilization dramatically enhanced binding to resin and recovery of the protein beyond expectation. Also pretreatment by Triton X-114 for removing endotoxin was effective for affinity chromatography. In chromatography performance, l-arginine was more effective than Triton X-114 treatment. Following Mono Q anion exchange chromatography, the target protein was isolated in high purity. The rhSCF protein specifically enhanced the viability of human myeloid leukemia cell line TF-1 and the proliferation and maturation of human mast cell line LAD2 cell. This novel protocol for the production of rhSCF is a simple, suitable, and efficient method.

Bacillus subtilis class Ib ribonucleotide reductase: high activity and dynamic subunit interactions.

The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (ß) subunits and determined to have a dimanganic-tyrosyl radical (Mn(III)2-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn(III)2-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min(-1) mg(-1)). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that B. subtilis RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an "active" RNR complex. We now report our efforts to optimize the activity of B. subtilis RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from Mn(III)2-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·/ß2. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ~1250 nmol min(-1) mg(-1) using a 1:1 mixture of NrdE:Mn(III)2-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (~1 µM), NrdE is a monomer (α) and Mn(III)2-Y· NrdF is a dimer (ß2). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations.

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: its role in arsenic detoxification.

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20mM and 15mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram+bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a kcat/KM value of 1.2×10(4)M(-1)s(-1). It also exhibited weak phosphatase activity with a kcat/KM value of 2.7×10(-4)M(-1)s(-1). The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural-functional characterization of a thermophilic arsenate reductase.

High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

High-level expression, purification and characterization of active human C1q and tumour necrosis factor-related protein-1 in Escherichia coli.

UNLABELLED: C1q and tumour necrosis factor-related proteins (CTRPs) are a family of adiponectin paralogues. CTRP1 plays important biological functions in diabetes, obesity and hypertension. To further explore the physiological roles of human CTRP1 and its mechanisms of action, hCTRP1 gene was expressed in Escherichia coli. In the E. coli expression system, a large amount of soluble thioredoxin (Trx)-hCTRP1 fusion protein could be produced using the expression plasmid pET32a (+) and induction with IPTG at 18°C, which accounts about 20% of the total soluble bacterial proteins. The recombinant Trx-hCTRP1 fusion protein was purified to an approx. 95% purity using Ni-NTA affinity chromatography and Superdex G-75 column with a yield of about 28-mg protein from 1-l bacterial cultures. The purified recombinant Trx-hCTRP1 was shown to be active under in vivo and in vitro assay conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: CTRP1 plays important biological functions and warrants further investigation. However, large-scale production of recombinant CTRP1 has been technically challenging, which becomes a major obstacle in the structural and functional analysis of this important family of proteins. To explore the possible clinical applications and mechanisms of its action, an efficient method to produce large amounts of active recombinant human CTRP1 is necessary. This study should facilitate basic functional and pharmacological studies of this important protein family.

Overproduction of a C5a receptor antagonist (C5aRA) in Escherichia coli.

The C5aR antagonist (C5aRA)(1), which blocks the interaction of C5a anaphylatoxin and its receptor C5aR, is one of the most potent therapeutic agents for the treatment of various autoimmune diseases and acute inflammatory conditions. Here we developed an efficient C5aRA production system using Escherichia coli. To produce functional C5aRA, which contains three disulfide bonds, we used E. coli Origami (DE3), which possessed an oxidative cytoplasm, as the production host. To improve solubility and ease in purification, we examined the effectiveness of three different fusion partners, including N utilization substrate A (NusA), maltose-binding protein (MBP), and thioredoxin A (TrxA), as well as three different culture temperatures (i.e., 25, 30, and 37°C). Among the three fusion partners, MBP exhibited the highest solubility in the fusion protein at all tested temperatures. However, the highest biological activity against C5aR was observed with the NusA fusion. For large-scale production, batch fermentation was also performed using a NusA-fused C5aRA production system by using a lab-scale bioreactor. After a 12-h cultivation, approximately 496mg/L of NusA-fused C5aRA could be produced.

Molecular cloning, expression, purification and characterization of thioredoxin from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178.

Thioredoxin (Trx) is a highly conserved and multi-functional protein that plays a pivotal role in maintaining the redox state of the cell and in protecting the cell against oxidative stress. Trx gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. AN178 was cloned and expressed as soluble protein in Escherichia coli (designated as PsTrx). Trx gene consisted of an open reading frame of 324-bp nucleotides encoding a protein of 108 amino acids with a calculated molecular mass of 11.88 kDa. The deduced protein included the conserved Cys-Gly-Pro-Cys active-site sequence. After purification by a single step Ni-NTA affinity chromatography, recombinant PsTrx with a high specific activity of 96.67 U/mg was obtained. The purified PsTrx had an optimal temperature and pH of 25 °C and 7.0, respectively, and showed about 55 % of the residual catalytic activity even at 0-10 °C. It had high tolerance to a wide range of NaCl concentrations (0-2 M NaCl) and was stable in the presence of H2O2. This research suggested that PsTrx displayed unique catalytic properties.