Structural and functional characterization of Solanum tuberosum VDAC36.

As it forms water-filled channel in the mitochondria outer membrane and diffuses essential metabolites such as NADH and ATP, the voltage-dependent anion channel (VDAC) protein family plays a central role in all eukaryotic cells. In comparison with their mammalian homologues, little is known about the structural and functional properties of plant VDACs. In the present contribution, one of the two VDACs isoforms of Solanum tuberosum, stVDAC36, has been successfully overexpressed and refolded by an in-house method, as demonstrated by the information on its secondary and tertiary structure gathered from circular dichroism and intrinsic fluorescence. Cross-linking and molecular modeling studies have evidenced the presence of dimers and tetramers, and they suggest the formation of an intermolecular disulfide bond between two stVDAC36 monomers. The pore-forming activity was also assessed by liposome swelling assays, indicating a typical pore diameter between 2.0 and 2.7 nm. Finally, insights about the ATP binding inside the pore are given by docking studies and electrostatic calculations.

Role of Hsp90 and ATP in modulating apyrase activity and firefly luciferase kinetics.

The present manuscript describes a novel bioassay consisting of apyrase and heat shock protein 90 (Hsp90) without additional co-chaperone supplementation; intended for high-throughput screening of anti-cancer drugs and prognosis of stress. In this regard, Hsp90 and adenosine 5'-triphosphate (ATP) mediated firefly luciferase (FLuc) kinetics was investigated using apyrase and FLuc as client proteins. Bioluminescent assay containing Hsp90, ATP, and apyrase led to complete loss of luminescence at 50 °C which indicates the protective role of Hsp90 against thermal denaturation. Similarly, the assay sample comprising Hsp90, ATP, and FLuc showed 2 fold increments in luminescence than their counterparts. Introduction of bovine serum albumin (BSA) to the pre-incubated assay mixture led to an initial rise in the luminescence (28%) in comparison to the sample containing Hsp90, ATP and FLuc. Therefore, FLuc based HTS assays are not suitable for clinical samples which may contain stabilizing agents. However, thermally denatured FLuc and apyrase could not regain their active conformation even when Hsp90 and ATP were introduced in the assay system. This observation justifies the role of Hsp90 to be protective rather than a reparation agent when acts without co-chaperones.

A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli.

An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time from the Escherichia coli expression system using short Strep-tag II (ST II). The recHPL-ST II was solubilized using 8 M urea from E.coli lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses in the presence of glycerol and Ca2+ for 2 days followed by gel filtration, 1.8-6 mg of active recHPL-ST II was obtained from 1 L of culture. The recHPL was non-glycosylated, but showed almost equal specific activity, pH-dependency and time-dependent stability compared to those of native porcine pancreatic lipase (PPL) at 37°C. However, the recHPL lost its lipolytic activity above 50°C, showing a lower heat-stability than that of native PPL, which retained half its activity at this temperature.

Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity.

Nicotinamide N-methyltransferase (NNMT) is an important biotransforming enzyme that catalyzes the transfer of a labile methyl group from the ubiquitous cofactor S-5'-adenosyl-l-methionine (SAM) to endogenous and exogenous small molecules to form methylated end products. NNMT has been implicated in a number of chronic disease conditions, including metabolic disorders, cardiovascular disease, cancer, osteoarthritis, kidney disease, and Parkinson's disease. We have developed a novel noncoupled fluorescence-based methyltransferase assay that allows direct ultrasensitive real-time detection of the NNMT reaction product 1-methylquinolinium. This is the first assay reported to date to utilize fluorescence spectroscopy to directly monitor NNMT product formation and activity in real time. This assay provided accurate kinetic data that allowed detailed comparative analysis of the NNMT reaction mechanism and kinetic parameters. A reaction model based on a random bireactant mechanism produced global curve fits that were most consistent with steady-state initial velocity data collected across an array of substrate concentrations. On the basis of the reaction mechanism, each substrate could independently bind to the NNMT apoenzyme; however, both substrates bound to the complementary binary complexes with an affinity ∼20-fold stronger compared to their binding to the apoenzyme. This reaction mechanism implies either substrate-induced conformational changes or bireactant intermolecular interactions may stabilize the binding of the substrate to the binary complex and formation of the ternary complex. Importantly, this assay could rapidly generate concentration response curves for known NNMT inhibitors, suggesting its applicability for high-throughput screening of chemical libraries to identify novel NNMT inhibitors. Furthermore, our novel assay potentially offers a robust detection technology for use in SAM substrate competition assays for the discovery and development of SAM-dependent methyltransferase inhibitors.

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes.

BACKGROUND: Manganese peroxidase (MnP) from Irpex lacteus F17 has been shown to have a strong ability to degrade recalcitrant aromatic pollutants. In this study, a recombinant MnP from I. lacteus F17 was expressed in Escherichia coli Rosetta (DE3) in the form of inclusion bodies, which were refolded to achieve an active enzyme. Further, we optimized the in vitro refolding conditions to increase the recovery yield of the recombinant protein production. Additionally, we attempted to express recombinant MnP in soluble form in E. coli, and compared its activity with that of refolded MnP. RESULTS: Refolded MnP was obtained by optimizing the in vitro refolding conditions, and soluble MnP was produced in the presence of four additives, TritonX-100, Tween-80, ethanol, and glycerol, through incubation at 16 °C. Hemin and Ca2+ supplementation was crucial for the activity of the recombinant protein. Compared with refolded MnP, soluble MnP showed low catalytic efficiencies for Mn2+ and H2O2 substrates, but the two enzymes had an identical, broad range substrate specificity, and the ability to decolorize azo dyes. Furthermore, their enzymatic spectral characteristics were analysed by circular dichroism (CD), electronic absorption spectrum (UV-VIS), fluorescence and Raman spectra, indicating the differences in protein conformation between soluble and refolded MnP. Subsequently, size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses demonstrated that refolded MnP was a good monomer in solution, while soluble MnP predominantly existed in the oligomeric status. CONCLUSIONS: Our results showed that two forms of recombinant MnP could be expressed in E. coli by varying the culture conditions during protein expression.

Generation of recombinant destabilase-lysozyme from medicinal leeches in three different expression systems.

Destabilase-lysozyme (mlDL) is an enzyme secreted by the salivary gland cells of medicinal leeches. Destabilase-lysozyme possesses lysozyme and isopeptidase activities. We generated recombinant destabilase-lysozyme isoform 2 in three expression systems, i.e., in the bacteria Escherichia coli, in the yeast Pichia pastoris, and in the human cell line Expi293F. In E. coli, we generated both polypeptide in inclusion bodies that was later undergone to the refolding and soluble protein that had been fused with the chaperone SlyD. The chaperone was later cleaved by a specific TEV-protease. In cultures of the yeast P. pastoris and the human cell line Expi293F, the soluble form of destabilase-lysozyme was accumulated in the culture media. For the generated enzymes, we determined the lysozyme, isopeptidase and fibrinolytic activities and tested their general antimicrobial effects. The comparisons of the enzymes generated in the different expression systems revealed that all of the destabilase-lysozymes obtained in the soluble forms possessed equal levels of lysozyme, isopeptidase and fibrinolytic activities that exceeded several to ten times the levels of the same activities of the destabilase-lysozyme renaturated from the inclusion bodies. A similar pattern of the differences in the levels of the general antimicrobial effects was observed for the destabilase-lysozymes generated in the soluble form and as inclusion bodies.

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. revealed by de novo transcriptome analysis.

Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.

Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry.

Crystal structure of the Mycobacterium tuberculosis phosphate binding protein PstS3.

Mycobacterium tuberculosis evades host immune responses by colonizing macrophages. Intraphagosomal M. tuberculosis is exposed to environmental stresses such as reactive oxygen and nitrogen intermediates as well as acid shock and inorganic phosphate (Pi) depletion. Experimental evidence suggests that expression levels of mycobacterial protein PstS3 (Rv0928) are significantly increased when M. tuberculosis bacilli are exposed to Pi starvation. Hence, PstS3 may be important for survival of Mtb in conditions where there is limited supply of Pi. We report here the structure of PstS3 from M. tuberculosis at 2.3-Å resolution. The protein presents a structure typical for ABC phosphate transfer receptors. Comparison with its cognate receptor PstS1 showed a different pattern distribution of surface charges in proximity to the Pi recognition site, suggesting complementary roles of the two proteins in Pi uptake.

Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein.

Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts.

The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of protein-protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368-606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH4)2SO4], sodium sulfate (Na2SO4), sodium chloride (NaCl), and ammonium chloride (NH4Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH4)2SO4 was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28-aa region (aa368-395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu³7², Leu³75, and Leu³95 of p239 was found to be critical for particle assembly, which was revealed by site-directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions.

Expression, high-pressure refolding and purification of human leukocyte cell-derived chemotaxin 2 (LECT2).

Human leukocyte cell-derived chemotaxin 2 (LECT2) is a chemotactic factor for neutrophils and a 16-kDa secreted protein consisting of 133 amino acids with three intramolecular disulfide bonds. Here, we propose an efficient method for the preparation of human LECT2 using a high hydrostatic pressure (HHP, 200 MPa) refolding technique. When LECT2 was over-expressed in Escherichia coli cells, most of LECT2 molecules became insoluble inclusion bodies (IBs). HHP was applied to the refolding of LECT2 from insoluble IBs, which dramatically improved the yield of the active LECT2. CD and NMR measurements demonstrated that the refolded LECT2 had a tertiary structure indistinguishable from the solubly expressed LECT2. In addition, both the refolded and solubly expressed LECT2 showed the same level of chemotactic activity.

Efficient overexpression and purification of active full-length human transcription factor Yin Yang 1 in Escherichia coli.

The Yin Yang 1 protein is a zinc finger transcription factor involved in the regulation of diverse cellular processes through DNA and protein-protein interactions. Here we present an improved method for the expression and purification of the human full-length YY1 protein from Escherichia coli. The protein was first purified using denaturing conditions, refolded using optimized conditions and then purified using a DNA-affinity column to ≥ 95% purity; this process provided a high final yield and highly active protein. The protein was active in EMSA and the fluorescence anisotropy assays. The protein retained its full activity and its initial concentration for several months when stored at -80° C. Thus, we have obtained YY1 protein with levels of activity and concentration that are suitable for spectroscopic and other biochemical studies.