Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography.

A scheme is described for the large scale purification of thioredoxin, thioredoxin reductase, and glutathione reductase. The scheme is based on an initial separation of thioredoxin from the two reductases by affinity chromatography on agarose-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (agarose-2',5'-ADP). The two reductases were then separated by hydrophobic chromatography and purified separately to homogeneity. Thioredoxin was purified to homogeneity by immunoadsorption to agarose containing immobilized goat anti-thioredoxin. Overall yields for thioredoxin, thioredoxin reductase, and glutathione reductase exceeded 80% in each case. Both reductases exhibit an absorption band at approximately 320 nm which appears due to a residual amount of tightly bound NADP. Presence of this absorption band has no apparent effect on the specific activity of either enzyme.     

Electrophoretic affinity chromatography: method validation

A new method for preparative-scale separation of biomolecules, electrophoretic affinity chromatography (EAC), is proposed in this paper. Separation by EAC is carried out in a long and ribbon-like multicompartment electrolyser separated by membranes, in which the two central compartments are used for packing the gel matrix and for sample loading respectively. Next to the central compartments are the elution compartments and electrode compartments. The electric field is applied perpendicular to the fluid flow in the compartments. Adsorption and desorption steps may both be carried out in the presence of an electric field, which transports the target components into the gel compartment for adsorption and the impurities into the elution compartments for washing. After the adsorption step an elution solution is introduced and the product is released from the gel matrix and washed out. Separation of human serum albumin (HSA) from human serum gives HSA product of high purity, as demonstrated by isoelectric focusing analysis. The characteristics of electrophoretic binding of HSA on Blue Sepharose Fast Flow are examined. The preliminary results show that this new method has advantages in terms of high rate of mass transfer and ease of scaling up, which are of particular interest when large-scale separation of biomolecules is considered.     

Thioredoxin and thioredoxin binding protein 2 in the liver

Thioredoxin (TRX) is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys- and constitutes a major thiol reducing system. TRX protects cells from stress-induced damage through antioxidative, antiapoptotic, and anti-inflammatory effect. In animal models, thioacetamide (TAA)-induced acute hepatitis and TAA-induced liver fibrosis was attenuated in TRX transgenic (TRXTG) mice. Plasma level of TRX is a good marker for hepatitis and nonalcoholic steatohepatitis (NASH) in human patients. Recently, we identified TRX binding protein 2 (TBP2) in a yeast two-hybrid screening. TBP2 regulates both the expression and reducing activity of TRX as well as cell growth. TBP2 knockout (TBP2KO) mice showed disorder in lipid metabolism. TBP2 plays a multiple role on cell growth and lipid and glucose metabolism. Thus, TRX and TBP2 play important roles in the pathophysiology of liver diseases, including NASH, indicating that ratio of TRX and TBP2 expression could be a novel marker of liver diseases like NASH.     

Floquet theory: a useful tool for understanding nonequilibrium dynamics

Many ecological systems experience periodic variability. Theoretical investigation of population and community dynamics in periodic environments has been hampered by the lack of mathematical tools relative to equilibrium systems. Here, I describe one such mathematical tool that has been rarely used in the ecological literature but has widespread use: Floquet theory. Floquet theory is the study of the stability of linear periodic systems in continuous time. Floquet exponents/multipliers are analogous to the eigenvalues of Jacobian matrices of equilibrium points. In this paper, I describe the general theory, then give examples to illustrate some of its uses: it defines fitness of structured populations, it can be used for invasion criteria in models of competition, and it can test the stability of limit cycle solutions. I also provide computer code to calculate Floquet exponents and multipliers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ribonuclease from cobra snake venom: purification by affinity chromatography and further characterization

A ribonuclease from cobra snake venom was isolated and purified to homogeneity using antibody-affinity chromatography, increasing the yield fourfold. The purified enzyme showed cytidylic acid specificity, as reported earlier. Further, the effects of temperature, pH, metal ions, inhibitors, and urea on the enzyme activity were studied. Snake venom RNase exhibited salt-dependent reversible association-dissociation behaviour. Immunological studies indicate that this enzyme shares one of the antigenic sites of RNase A. The partial N-terminal sequence of the enzyme showed considerable homology with phospholipases from snake venom; however, the enzyme itself did not show any phospholipase activity.     

Thioredoxin system in obligate anaerobe Desulfovibrio desulfuricans: Identification and characterization of a novel thioredoxin 2

Metal corroding sulfate reducing bacteria have been poorly characterized at molecular level due to difficulties pertaining to isolation and handling of anaerobes. We report here for the first time the presence and characterization of thioredoxin 2 in an obligate anaerobic dissimilatory sulfate reducing bacterium Desulfovibrio desulfuricans. In silico analysis of the D. desulfuricans genome revealed the presence of thioredoxin 1 (dstrx1), thioredoxin 2 (dstrx2) and thioredoxin reductase (dstrxR) genes. These genes were found to be actively expressed by the bacteria under the anaerobic growth conditions. We have overexpressed the anaerobic thioredoxin genes in E. coli to produce functionally active recombinant proteins. Recombinant DsTrxR recognized both DsTrx1 and DsTrx2 as its substrate. Mutation studies revealed that the activity of DsTrx2 can be completely abolished with a single amino acid mutation (C69A) in the signature motif 'WCGPC'. Furthermore, the N-terminal domain of DsTrx2 containing two extra CXXC motifs was found to have a negative regulation on its biochemical activity. In conclusion, we have shown the presence of thioredoxin 2 for the first time in an obligate anaerobe which in this anaerobe may be required for its survival under either oxidative stress conditions or metal ion hemostasis.     

Murine C4-binding protein: a rapid purification method by affinity chromatography

A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.     

Thioredoxin and thioredoxin reductase: Current research with special reference to human disease

Thioredoxin (Trx) and thioredoxin reductase (TrxR) plus NADPH, comprising the thioredoxin system, has a large number of functions in DNA synthesis, defense against oxidative stress and apoptosis or redox signaling with reference to many diseases. All three isoenzymes of mammalian TrxR contain an essential selenocysteine residue, which is the target of several drugs in cancer treatment or mercury intoxication. The cytosolic Trx1 acting as the cells’ protein disulfide reductase is itself reversibly redox regulated via three structural Cys residues. The evolution of mammalian Trx system compared to its prokaryotic counterparts may be an adaptation to the use of hydrogen peroxide and nitric oxide in redox regulation and signal transduction.     

A new method for the screening of solid-phase combinatorial libraries for affinity chromatography

A new methodology for the rapid assessment of affinity ligands synthesized by combinatorial solid-phase chemistry is reported. This screening strategy utilizes the target protein conjugated to FITC, and represents an almost universal technique for the preliminary screening of solid-phase combinatorial libraries. The assessment of a triazine-scaffolded solid-phase combinatorial library of ligands, designed to bind to human IgG, was performed with FITC-human IgG, and the results compared with those obtained by conventional affinity chromatographic screening assays. The effect of different molar conjugation ratios of FITC-IgG (F/P) was evaluated. Independently of the F/P ratio, no false negative results were observed, although lower F/P ratios diminished non-specific interactions and the number of false positives. The nature of the substituents on the triazine scaffold was not related to the number of false positive IgG-binding ligands. The reproducibility of the FITC technique, using FITC-human IgG conjugates with low F/P ratio (F/P=2), was also evaluated. The FITC-based technique proved to be efficient and accurate in the identification of strongly binding ligands (binding >50% of loaded protein, by standard affinity chromatographic assays), and is envisaged as a versatile and cost-effective method to screen other systems, and evaluate several binding/elution conditions at small-scale, prior to scale-up to standard affinity chromatography.     

Flux-sum analysis: a metabolite-centric approach for understanding the metabolic network

Background  

Constraint-based flux analysis of metabolic network model quantifies the reaction flux distribution to characterize the state of cellular metabolism. However, metabolites are key players in the metabolic network and the current reaction-centric approach may not account for the effect of metabolite perturbation on the cellular physiology due to the inherent limitation in model formulation. Thus, it would be practical to incorporate the metabolite states into the model for the analysis of the network.     

Thioredoxin is essential for photosynthetic growth. The thioredoxin m gene of Anacystis nidulans

We have taken advantage of the transformation properties of the cyanobacterium Anacystis nidulans R2 to investigate the importance of thioredoxin for photosynthetic growth. The gene encoding thioredoxin m, designated trxM, was cloned from A. nidulans using a synthetic oligonucleotide probe. Based on the nucleotide sequence, thioredoxin m of A. nidulans is composed of 107 amino acids and shares 84, 48, and 48% sequence identity with thioredoxins from Anabaena, spinach, and Escherichia coli, respectively. The trxM gene is single copy and is transcribed on a 510-nucleotide mRNA. We demonstrate that disruption of the trxM gene with a kanamycin resistance gene cartridge is a lethal mutation. Although dispensable in E. coli, thioredoxin is essential for the photosynthetic growth of A. nidulans.     

Improved method for removal of albumin from serum by affinity chromatography

Ges prepared from alkyl succinic anhydride coupled to agarose beads by diaminoalkane spacers have been studied to evaluate the influence of the chain length of both the alkyl succinic anhydride and the spacer on the gel's quantitative capacity and specificity to absorb albumin. The maximum absorptive eapacity for albumin of these gels varied from 13 to 30 mg of albumin/ml of gel and was related to the chain length of the alkyl succinic anhydride and the spacer. Before gel capacities were reached, eluates were albumin free when examined by electroimmunoassay (sensitivity, 1 μg/ml). The gels were not completely specific for albumin. However, pretreatment of the gels with gelatin decreased their nonspecific binding.     

Concanavalin a affinity chromatography for efficient baculovirus purification

Baculovirus has emerged as a novel gene delivery and vaccine vector, and the demand for purified baculovirus is rising due to the increasing in vivo applications. Since the baculoviral envelope protein gp64 is a glycoprotein, we aimed to develop a concanavalin A (Con A) chromatography process, which harnessed the possible affinity interaction between gp64 and Con A, for simple and effective baculovirus purification. Throughout the purification process the virus stability and recovery were assessed by quantifying the virus transducing titers [TT, defined as transducing units (TU) per milliliter] and viral particles (VP). We found that baculovirus stability was sensitive to buffer conditions and diafiltration with a tangential flow filtration system LabScale using 300 K membranes yielded recoveries of ≈75% in TT and 82% in VP. The diafiltered baculovirus strongly bound to the Con A column as evidenced by the low virus losses to the flow through and wash fractions. The wash steps eliminated >99% of protein impurities and elution with 0.6 M α‐D ‐methylmannoside at room temperature led to the recoveries of ≈16% in VP and ≈15.3% in TU. The resultant VP/TU ratio was as low as 41.4, attesting the high quality of the purified virus. Further elution with 1 M α‐D ‐methylmannoside recovered another 6% virus TU, yielding a cumulative recovery of ≈21.3% in TU. These data demonstrated for the first time that Con A chromatography is suitable for baculovirus purification, and may be used for the purification of other viruses with surface glycoproteins. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Use of an affinity chromatography method for isolating monospecific Salmonella antibodies

Obtaining antibodies to individual components of Salmonella antigenic complex is highly important for investigations aimed at the study of the antigenic structure of bacteria, their serological identification and the development of diagnostic preparations. The method of obtaining antibodies by the oxidation of Salmonella antigens with sodium periodate and creating immunosorbents based on these antibodies with subsequent affinity chromatography has been developed. Monospecific antibodies thus obtained (O2, O4, O9) have been studied and used as monospecific preparations in the agglutination test, the immunofluorescence test and the immunosorbent assay. The development of methods for stabilizing these preparations, thus ensuring their wide practical use, may be of interest.     

A method for the purification of histone fraction F3 by affinity chromatography

Calf thymus histone fraction F3 has been purified in high yield by affinity chromatography. The protein is attached reversibly to an organomercurial—Sepharose—by the thiol groups of its cysteinyl residues, while all other histones lacking -SH groups pass through the column. Histone F3 is subsequently eluted with -SH reagents of low molecular weight, such as cysteine or β-mercaptoethanol. The purified F3 shows electrophoretic heterogeneity due to different degrees of acetylation of the parent polypeptide chain. Under oxidizing conditions it forms a complex mixture of dimers and oligomers, all of which have some available -SH groups.