Two-step Chromatographic Procedure Research Articles

Expression, characterization and cytotoxicity of recombinant l-asparaginase II from Salmonella paratyphi cloned in Escherichia coli

L-asparaginase is a remarkable antineoplastic enzyme used in medicine for the treatment of acute lymphoblastic leukemia (ALL) as well as in food industries. In this work, the L-asparaginase-II gene from Salmonella paratyphi was codon-optimized, cloned, and expressed in E. coli as a His-tag fusion protein. Then, using a two-step chromatographic procedure it was purified to homogeneity as confirmed by SDS-PAGE, which also showed its monomeric molecular weight to be 37 kDa. This recombinant L-asparaginase II from Salmonella paratyphi (recSalA) was optimally active at pH 7.0 and 40 °C temperature. It was highly specific for L-asparagine as a substrate, while its glutaminase activity was low. The specific activity was found to be 197 U/mg and the kinetics elements Km, Vmax, and kcat were determined to be 21 mM, 28 μM/min, and 39.6 S−1, respectively. Thermal stability was assessed using a spectrofluorometer and showed Tm value of 45 °C. The in-vitro effects of recombinant asparaginase on three different human cancerous cell lines (MCF7, A549 and Hep-2) by MTT assay showed remarkable anti-proliferative activity. Moreover, recSalA exhibited significant morphological changes in cancer cells and IC50 values ranged from 28 to 45.5 μg/ml for tested cell lines. To investigate the binding mechanism of SalA, both substrates L-asparagine and l-glutamine were docked with the protein and the binding energy was calculated to be −4.2 kcal mol−1 and − 4.4 kcal mol−1, respectively. In summary, recSalA has significant efficacy as an anticancer agent with potential implications in oncology while its in-vivo validation needs further investigation.

High purity of human secreted phospholipase A2 group IIE in Pichia pastoris using basal salts medium comparison with YPD medium

Secreted phospholipase A2s (sPLA2s) are a group of enzymes with 6–8 disulfide bonds that participate in numerous physiological processes by catalyzing the hydrolysis of phospholipids at the sn-2 position. Due to their high content of disulfide bonds and hydrolytic activity toward cell membranes, obtaining the protein of sPLA2s in the soluble and active form is challenging, which hampers their functional study. In this study, one member of recombinant human sPLA2s, tag-free group IIE (GIIE), was expressed in Pichia pastoris. The protein GIIE was purified from the crude culture supernatant by a two-step chromatography procedure, a combination of cation exchange and size-exclusion chromatography. In the shake flask fermentation, Protein of GIIE with higher purity was successfully obtained, using basal salts medium (BSM) instead of YPD medium. In the large-scale fermentation, each liter of BSM produced a final yield of 1.2 mg pure protein GIIE. This protocol will facilitate further research of GIIE and provide references for the production of other sPLA2 members.

Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

Hydra actinoporin-like toxin-1 (HALT-1) has been isolated from Hydra magnipapillata and is highly cytolytic against various human cells including erythrocyte. Previously, recombinant HALT-1 (rHALT-1) was expressed in Escherichia coli and purified by the nickel affinity chromatography. In this study, we improved the purification of rHALT-1 by two-step purifications. Bacterial cell lysate containing rHALT-1 was subjected to the sulphopropyl (SP) cation exchange chromatography with different buffers, pHs, and NaCl concentrations. The results indicated that both phosphate and acetate buffers facilitated the strong binding of rHALT-1 to SP resins, and the buffers containing 150 mM and 200 mM NaCl, respectively, removed protein impurities but retain most rHALT-1 in the column. When combining the nickel affinity chromatography and the SP cation exchange chromatography, the purity of rHALT-1 was highly enhanced. In subsequent cytotoxicity assays, 50% of cells could be lysed at ∼18 and ∼22 µg/mL of rHALT-1 purified with phosphate and acetate buffers, respectively.•HALT-1 is a soluble α-pore-forming toxin of 18.38 kDa.•rHALT-1 was purified by nickel affinity chromatography followed by SP cation exchange chromatography.•The cytotoxicity of purified rHALT-1 using 2-step purifications via either phosphate or acetate buffer was comparable to those previously reported.

Safe Sialidase Production by the Saprophyte Oerskovia paurometabola: Gene Sequence and Enzyme Purification.

Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed β-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application.

A Simple Micropreparative Gel Electrophoresis Technique for Purification of Nanoscale Materials

Many biochemical, biomedical, and material applications hinge on the ability to effectively separate and purify nanoscale materials. Though this need is largely addressed with biological macromolecules using a variety of chromatographic and electrophoretic purification techniques, such techniques are usually laborious, time-consuming, and often require complex and costly instalments that are inaccessible to most laboratories. Synthetic nanoparticles face similar purification challenges, often relying on techniques that are material-specific. In this work, we introduce a versatile micro-preparative (MP) method based on polyacrylamide gel electrophoresis (PAGE) to purify biological samples containing proteins, nucleic acids, and complex bioconjugates, as well as synthetic nanoparticles based on graphene quantum dots (GQDs). Using a conventional vertical slab PAGE, we demonstrate the extraction of purified DNA, proteins, and DNA-protein bioconjugates from their respective mixtures using MP-PAGE. We apply this system to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using specialized commercial devices. We also demonstrate the purification of folded enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, comparable to purities achieved using a two-step size exclusion and immobilized metal-ion affinity chromatography purification procedure. Moreover, we demonstrate the successful isolation of an EYFP-DNA bioconjugate that otherwise could not be processed using the two-step chromatography procedure. Finally, the technique was further extended to demonstrate size-dependent separation of a commercial mixture of GQDs into three different fractions with distinct optical properties. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

Expression, purification and characterization of the transcription termination factor Rho from Azospirillum brasilense

The Transcription Termination factor Rho is a ring-shaped, homohexameric protein that causes transcript termination by interaction with specific sites on nascent mRNAs. The process of transcription termination is essential for proper expression and regulation of bacterial genes. Although Rho has been extensively studied in the model bacteria Escherichia coli (EcRho), the properties of other Rho orthologues in other bacteria are poorly characterized. Here we present the heterologous expression and purification of untagged Rho protein from the diazotrophic environmental bacterium Azospirillum brasilense (AbRho). The AbRho protein was purified to >99% through a simple, reproducible and efficient purification protocol, a two-step chromatography procedure (affinity/gel filtration). By using analytical gel filtration and dynamic light scattering (DLS), we found that AbRho is arranged as an homohexamer as observed in the EcRho orthologue. Secondary structure and enzyme activity of AbRho was also evaluate indicating a properly folded and active protein after purification. Enzymatic assays indicate that AbRho is a RNA-dependent NTPase enzyme.

Crystal structure of glutamate dehydrogenase 2, a positively selected novel human enzyme involved in brain biology and cancer pathophysiology.

Mammalian glutamate dehydrogenase (hGDH1 in human cells) interconverts glutamate to α-ketoglutarate and ammonia while reducing NAD(P) to NAD(P)H. During primate evolution, humans and great apes have acquired hGDH2, an isoenzyme that underwent rapid evolutionary adaptation concomitantly with brain expansion, thereby acquiring unique catalytic and regulatory properties that permitted its function under conditions inhibitory to its ancestor hGDH1. Although the 3D-structures of GDHs, including hGDH1, have been determined, attempts to determine the hGDH2 structure were until recently unsuccessful. Comparison of the hGDH1/hGDH2 structures would enable a detailed understanding of their evolutionary differences. This work aimed at the determination of the hGDH2 crystal structure and the analysis of its functional implications. Recombinant hGDH2 was produced in the Spodoptera frugiperda ovarian cell line Sf21, using the Baculovirus expression system. Purification was achieved via a two-step chromatography procedure. hGDH2 was crystallized, X-ray diffraction data were collected using synchrotron radiation and the structure was determined by molecular replacement. The hGDH2 structure is reported at a resolution of 2.9Å. The enzyme adopts a novel semi-closed conformation, which is an intermediate between known open and closed GDH1 conformations, differing from both. The structure enabled us to dissect previously reported biochemical findings and to structurally interpret the effects of evolutionary amino acid substitutions, including Arg470His, on ADP affinity. In conclusion, our data provide insights into the structural basis of hGDH2 properties, the functional evolution of hGDH isoenzymes, and open new prospects for drug design, especially for cancer therapeutics.

New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom.

Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80–6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bβ chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

Regulation of glutamate dehydrogenase (GDH) in response to whole body freezing in wood frog liver linked to differential acetylation and ADP-ribosylation

Glutamate dehydrogenase (GDH) represents a critical enzyme catalyzing the reaction spanning amino acid catabolism, the Krebs cycle, and urea production in the wood frog. GDH breaks down glutamate and NAD+ to generate α-ketoglutaric acid (α-KG), NADH and ammonium that can be metabolized to form urea. Purification of GDH from control and frozen male wood frog livers was performed using a two-step column chromatography procedure with a cation exchange column and a GTP-agarose affinity column. Analysis of kinetic parameters of the purified GDH showed several notable differences between the control and stress. Under standard assay conditions, the affinity of GDH for its substrates was significantly higher for the freeze-exposed enzyme than for the control (glutamate Km: 41% decrease, NAD+ Km: 40% decrease). The maximal activity for the control enzyme was also noted to be lower than the frozen. This suggests that the frozen form of the GDH was activated relative to the control form. Western blot analysis of common posttranslational modifications indicated that the frozen enzyme had a lower degree of acetylation and ADP-ribosylation than its control counterpart. These results suggest that GDH is regulated in the wood frog liver by means of altering post-translational modifications in response to freezing.

Heterologous Expression, Purification and Immunoreactivity of the Antigen 5 from Polybia paulista Wasp Venom.

Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.

Expression and Purification of Biologically Active Human Bone Morphogenetic Protein-4 in Recombinant Chinese Hamster Ovary Cells.

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of EC50 of 2.93 ng/ml.

A New Two-Step Chromatographic Procedure for Fractionation of Potato Proteins with Potato Fruit Juice and Spray-Dried Protein as Source Materials

In an effort to purify potato proteins of superior food grade quality, a new procedure involving anion exchange (ion exchange (IEX)) and hydrophobic interaction chromatography (HIC) was established. Liquid potato fruit juice (PFJ) or re-suspended spray-dried protein was separated by IEX yielding two fractions: a protease inhibitor (PI)-rich fraction and a patatin-rich fraction. Each of these fractions was re-chromatographed on HIC, resulting in two new sub-fractions which were characterised by electrophoresis and mass spectrometry. A high-quality powder should have high lightness, low polyphenol oxidase (PPO) activity and glycoalkaloid content below 150 μg/g. The PI fraction from spray-dried powder had high lightness (L* = 83) compared to patatin (L* = 50), whereas IEX purification from PFJ resulted in a PI fraction with decreased lightness (L* = 66) and a patatin fraction with increased lightness (L* = 68). HIC fractionation led to increased lightness for patatin fraction, but decreased lightness for the PFJ PI fractions. HIC purification significantly lowered PPO activity in the fractions due to its selective affinity. The lowest total glycoalkaloid content was generally found in HIC fractions from spray-dried powder (150 μg/g), while high levels (>1000 μg/g) were found in PI fractions from PFJ. In conclusion, it was possible to obtain a PI-rich protein isolate from powder with good-quality attributes after both IEX and HIC, while for patatin, the best quality was obtained after the HIC only, and there, the colour or PPO activity may still be a problem depending on source material.

Development of long-acting ciliary neurotrophic factor by site-specific conjugation with different-sized polyethylene glycols and transferrin

To overcome the deficiency of rapid elimination from blood, the truncated human recombinant ciliary neurotrophic factor was formulated by site-specific attachment of different-sized PEG-maleimide or by cross-linking with human transferrin through a hetero-bi-functional PEG linker (NHS-PEG5k-MAL). The PEGylated CNTF was purified by a two-step chromatography procedure and the transferrin coupling CNTF conjugate was separated through an elegant protocol. The conjugation site on CNTF was identified by peptide mapping analysis and validated that the linkage of the conjugates was specifically happened to Cys17 residue. Although both PEGylated and transferrin coupling CNTF demonstrated decreased cell based residual activity, markedly enhanced pharmacokinetic behaviors in normal male Sprague-Dawley rats were observed, especially for the PEG40k-CNTF with approximately 58-times improvement compared with the unmodified counterpart. The evaluation of the in vivo potency of body weight-losing was performed with normal male C57BL6 mice and the results revealed that both PEGylation and transferrin coupling could achieve improved therapeutic benefits relative to that of CNTF. Besides, PEG20k/40k-CNTF demonstrated more effective than transferrin coupling CNTF (Tf-PEG5k-CNTF) despite that the later showed preferable pharmacokinetic profile and cell based residual activity compared with PEG20k-CNTF. Weekly subcutaneous administration of PEG40k-CNTF with 0.5mg/kg and 1.0mg/kg dose resulted in approximately 35% and 50% decrease in food intake during one interval period of injection, indicating that PEG40k-CNTF is the most potential anti-obese agent for therapeutics.

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells

ABSTRACTInsulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500 µg/L) and the half-life of IGF-1 in blood circulation is only 4.5 min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA–IGF-1 reached 100 mg/L. The fusion protein HSA–IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA–IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA–IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA–IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.

Production of human mutant biologically active hepatocyte growth factor in Chinese hamster ovary cells

ABSTRACTHepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12 mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins.

Atypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical α, β, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase.

Collagenase produced from Aspergillus sp. (UCP 1276) using chicken feather industrial residue.

An extracellular collagenolytic serine protease was purified from Aspergillus sp., isolated from the Caatinga biome in northeast Brazil by a two-step chromatographic procedure, using an anion-exchanger and gel filtration. The enzyme was produced by submerged fermentation of feather residue as a substrate. The purified collagenase showed a 2.09-fold increase in specific activity and 22.85% yield. The enzyme was a monomeric protein with a molecular mass of 28.7 kDa, estimated by an SDS-PAGE and AKTA system. The optimum temperature and pH for enzyme activity were around 40°C and pH 8.0, respectively. The enzyme was strongly inhibited by phenyl-methylsulfonyl fluoride, a serine protease inhibitor, and was thermostable until 65°C for 1 h. We then evaluated the enzyme's potential for degradation of Type I and Type V collagens for producing peptides with antifungal activity. Our results revealed that the cleavage of Type V collagen yielded more effective peptides than Type I, inhibiting growth of Aspergillus terreus, Aspergillus japonicus and Aspergillus parasiticus. Both groups of peptides (Type I and Type V) were identified by SDS-PAGE. To conclude, the thermostable collagenase we purified in this study has various potentially useful applications in the fields of biochemistry, biotechnology and biomedical sciences.

Novel haemoglobin-derived antimicrobial peptides from chicken (Gallus gallus) blood: purification, structural aspects and biological activity.

To purify and characterize antimicrobial peptides derived from the acid extract of Gallus gallus blood cells. Two polypeptides (i.e. CHb-1 and CHb-2) with antibacterial activity were detected in the acidic extract of blood cells from chicken (G.gallus). The isolated peptides that possessed a potent antibacterial activity were purified using a two-step chromatography procedure that involved solid-phase extraction of a total protein/peptide extract followed by thin fractionation by reversed-phase high performance liquid chromatography (RP-HPLC). The molecular masses of the purified peptides were similar and were 4824·4 and 4825·2Da, which have been measured by matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF MS). Their amino acid sequences were determined by Edman degradation and showed that the peptides were fully identical to the two fragments of G.gallus α-haemoglobin localized into different subunits (A and D respectively). The peptides were active in micromolar concentrations against Gram-negative Escherichia coli K12 TG1. Using the 1-N-phenylnaphthylamine, the FITC-dextran labelled probes and the live/dead staining allowed to show the hemocidin mode of action and estimate the pore size. In this study, for the first time, α-haemoglobin from chicken (G.gallus) has been investigated as a donor of the two high homologous native peptide fragments that possess potent antibacterial activity invitro. These are membrane-active peptides and their mechanism of action against E.coli involves a toroidal pore formation. The obtained results expand the perception of the role of haemoglobin in a living system, describing it as a source of multifunction substances. Additionally, the data presented in this paper may contribute to the development of new, cost-effective, antimicrobial agents.

Purification, preliminary X-ray crystallography and biophysical studies of triose phosphate isomerase-β-globin subunit complex

Triose phosphate isomerase (TIM) is a cytoplasmic enzyme of prime importance in the mammalian glycolytic pathway. It has a major role in the conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We have successfully purified a stable complex of TIM with β-globin subunit from the sheep kidney using a simple two-step chromatography procedure. It is seen for the first time that TIM is forming a stable complex with β-globin. The purified protein-protein complex was crystallized and preliminary diffraction data were collected at 2.1Å resolution. We further studied guanidinium chloride (GdmCl)-induced denaturation of TIM-β-globin complex by monitoring changes in the mean residue ellipticity at 222nm ([θ]222) and difference absorption coefficient at 406nm (Δε406) at pH 7.5 and 25°C. We have observed that GdmCl-induced denaturation is reversible. Coincidence of normalized transition curves of both physical properties ([θ]222 and Δε406) suggests that folding/unfolding of TIM and β-subunit proteins is a two-state process. Denaturation curves of [θ]222 and Δε406 were used to estimate the stability parameters of the protein-protein complex. This is the first report on the isolation, purification, crystallization and biophysical characterization of the naturally occurring complex of TIM with the β-globin subunit.

Fed-batch production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in soluble form in Escherichia coli and its purification and characterization

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.