8M Urea Research Articles

“Proteomineering” or not? The debate on biomarker discovery in sera continues

The performance of sera pre-treatments for biomarker discovery has been recently assessed as very poor not only for immuno-subtraction, in turn evaluated as a tool unable to look deep into the low-abundance proteome (LAP) and thus incapable to lead to any novel biomarker discovery (J Proteome Res 2010;9:4982-4991), but also for combinatorial peptide ligand libraries (CPLL) (Proteomics 2010;10:1416-1425). The performance of both tools has been given as enabling to barely detect a meagre 25% more as compared to control, untreated sera. Meanwhile, other studies indicated the extreme effectiveness of peptide libraries to enlarge the knowledge of proteome compositions. In this contradictory situation we are here re-evaluating some protocol aspects and report that indeed CPLL is an excellent tool, able to dig really deep into the low-abundance proteome. The problem is that in those reports under-optimized capture and elution protocols had been adopted. With the protocols here reported, namely (a) abandoning the step of adding 150mM salt to the sample; (b) capture at three different pH values (pH 4.0, 7.0 and 9.3) and (c), most importantly, eluting from CPLL beads in 4% boiling SDS in the presence of 25mM DTT, we can largely expand the windows of visibility. In particular, it is here shown that a common elution protocol adopted in several reports, in 4M urea and 1% CHAP, barely elutes about 15% of the captured species. Nevertheless if the CPLL beads thus treated are further eluted with boiling SDS-DTT, an additional 80% is recovered.

Multi-Scale Simulations of Proteins in Different Solvent Conditions

We developed a multiscale approach (MultiSCAAL) that integrates the potential of mean force (PMF) obtained from all-atomistic molecular dynamics simulations with a knowledge-based energy function for coarse-grained molecular simulation in better exploring the energy landscape of a small protein under chemical interference such as chemical denaturation. The two key features of this scheme are the Boltzmann inversion and a protein atomistic reconstruction method we previously developed (SCAAL). Using MultiSCAAL, we were able to enhance the sampling efficiency of proteins solvated by explicit water molecules. Our method has been tested on the folding energy landscape of a small protein Trp-cage with explicit solvent under 8M urea using both the all-atomistic replica exchange molecular dynamics (AA-REMD) and MultiSCAAL. We compared computational analyses on ensemble conformations of Trp-cage with its available experimental NOE distances. The analysis demonstrated that conformations explored by MultiSCAAL better agree with the ones probed in the experiments because it can effectively capture the changes in side chain orientations that can flip out of the hydrophobic pocket in the presence of urea and water molecules. In this regard, MultiSCAAL is a promising and effective sampling scheme for investigating chemical interference which presents a great challenge when modeling protein interactions in vivo.

Cloning, purification and characterization of the ribosomal protein L11 from E. coli

A high-expression system of L11 was constructed and investigated its interaction with other elements of the ribosome using physicochemical methods. The gene rplK, coding for the protein L11 from the E. coli 50S ribosomal subunit was amplifyied, cloned and over-expressed. The protein L11 was purified under native and denaturing conditions, refolded and the structure of both proteins was compared. The protein L11 properly refolded from 6M urea after dialysis. Experiments on binding of proteins L11, RRF and EF-G from Escherichia coli were performed by ana-lytical centrifugation and Biacore. Specific binding between protein L11 and RRF by analytical cen-trifugation was not detected probably due to struc-tural reasons. These findings may be helpful in the design of new antibiotics that specifically disrupt the interactions in the “GTP-associated site” of the bac-terial ribosome, as many of them are not effective anymore. A common intrinsically disordered region of protein L11 was found to be the amino acid se-quence 86-97, while the residues 67-74, containing the linker region, are predicted to be disordered by DisEMBL.

Mixed Hydrotropy Solubilization Approach for Quantitative Estimation of Eprosartan Mesylate and Hydrochlorthiazide by UV Spectrophotometer

Two simple, accurate, novel, safe and precise methods were developed for the simultaneous estimation of poorly water-soluble drugs Eprosartan Mesylate and Hydrochlorthiazide in tablet dosage form using 2M Sodium acetate and 8M Urea solution (50:50% W/V) as a mixed hydrotropic solution. Eprosartan Mesylate and Hydrochlorthiazide show maximum absorbances at 267.5 and 271.5 nm respectively. Sodium acetate and Urea solution did not show any absorbance above 240 nm and thus no interference in the estimation of drugs was seen. Eprosartan Mesylate and Hydrochlorthiazide follows the Beer’s law in the concentration range of 15-75 and 5-25 μg/ml (r 2 = 0.9994 and 0.9996). Method-A employs a simultaneous equation method using 267.5 and 271.5 nm as two analytical wavelengths, Method-B, an absorption ratio method, uses 271.5 and 277 nm as two analytical wavelengths for estimation of Eprosartan Mesylate and Hydrochlorthiazide. The optimized methods showed good reproducibility and recovery with ranging from 95.08±0.086 to 99.82±0.097 EPS and HCZ respectively. The developed methods were validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values therefore the both methods can be used for routine monitoring of EPS and HCZ in industry in the assay of bulk drug and tablets.

Escherichia coli-derived von Willebrand factor-A2 domain fluorescence/Förster resonance energy transfer proteins that quantify ADAMTS13 activity

The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET (fluorescence/Förster resonance energy transfer) proteins, where variants of yellow fluorescent protein (Venus) and cyan fluorescent protein (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, and 77 amino acids) of this domain. These proteins were expressed in Escherichia coli in soluble form, and they exhibited FRET properties. Results show that the introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13-mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30min. VWF-A2 conformation changed progressively, and not abruptly, on increasing urea concentrations. Although proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity and as a tool to study VWF-A2 conformation dynamics.

Isolation and fractionation of soil humin using alkaline urea and dimethylsulphoxide plus sulphuric acid

Humin, the most recalcitrant and abundant organic fraction of soils and of sediments, is a significant contributor to the stable carbon pool in soils and is important for the global carbon budget. It has significant resistance to transformations by microorganisms. Based on the classical operational definition, humin can include any humic-type substance that is not soluble in water at any pH. We demonstrate in this study how sequential exhaustive extractions with 0.1M sodium hydroxide (NaOH) + 6M urea, followed by dimethylsulphoxide (DMSO) + 6% (v/v) sulphuric acid (H(2)SO(4)) solvent systems, can extract 70-80% of the residual materials remaining after prior exhaustive extractions in neutral and aqueous basic media. Solid-state (13)C NMR spectra have shown that the components isolated in the base + urea system were compositionally similar to the humic and fulvic acid fractions isolated at pH12.6 in the aqueous media. The NMR spectra indicated that the major components isolated in the DMSO + H(2)SO(4) medium had aliphatic hydrocarbon associated with carboxyl functionalities and with lesser amounts of carbohydrate and peptide and minor amounts of lignin-derived components. The major components will have significant contributions from long-chain fatty acids, waxes, to cuticular materials. The isolates in the DMSO + H(2)SO(4) medium were compositionally similar to the organic components that resisted solvation and remained associated with the soil clays. It is concluded that the base + urea system released humic and fulvic acids held by hydrogen bonding or by entrapment within the humin matrix. The recalcitrant humin materials extracted in DMSO + H(2)SO(4) are largely biological molecules (from plants and the soil microbial population) that are likely to be protected from degradation by their hydrophobic moieties and by sorption on the soil clays. Thus, the major components of humin do not satisfy the classical definitions for humic substances which emphasise that these arise from microbial or chemical transformations in soils of the components of organic debris.

The major surface Vsp proteins of Brachyspira hyodysenteriae form antigenic protein complexes

The Vsp proteins are the major outer membrane proteins of Brachyspira hyodysenteriae, the causative agent of swine dysentery. Eight vsp genes have been identified in B. hyodysenteriae strain B204, arranged into two four-gene loci, and at least two of the corresponding proteins are produced in vitro. The aims of this study were to characterise the vsp genes of the virulent Australian B. hyodysenteriae strain X576 and their corresponding proteins, Genomic sequence comparison with strains B204 and WA1 demonstrated that the number of vsp genes varies between B. hyodysenteriae strains, although the chromosomal locations of the vsp gene loci are consistent. We identified two additional vsp-like genes, designated vspI and vspJ, in each of the three strains. Double SDS-PAGE was used to demonstrate that Vsp proteins of B. hyodysenteriae strain X576 form multimeric protein complexes in the outer membrane that are stable in 6M urea but dissociate after boiling. The Vsp complexes primarily consisted of VspF but also contain VspE and VspI. VspD was also found in a series of complexes slightly larger than the more abundant VspF complexes. Vsp proteins are purported to be antigenic; however little direct data are available to support this claim. In this study convalescent pig sera did not bind denatured Vsp proteins by Western blotting, but did bind the Vsp complexes on Western blots, showing that conformational epitopes may be important in immune recognition of these major outer membrane proteins. This is the first definitive demonstration of the antigenicity of these proteins in swine dysentery.

Sequential recognition of antigenic markers of Toxoplasma gondii tachyzoite by pooled sera of mice with experimental toxoplasmosis

Accurate diagnosis of maternal toxoplasmosis can enhance the success of medical treatment and prevent congenital transmission. The current diagnostic methods have many limits, and they poorly differentiate between recent and latent infections. The present work was conducted to record the sequential recognition of antigenic markers of both Toxoplasma tachyzoites whole extract and glycosylinositolphospholipids (GIPLs)-enriched fraction by specific IgG and IgM, respectively, by immunoblotting analysis of the antigens against daily pooled serum samples from mice with experimentally induced recent and latent toxoplasmosis. IgG avidity immunoblotting was tested by using a wash with 6M urea solution as antigen-antibody disrupting agent. Band of 10kDa reacted exclusively with low-avidity IgG in pooled sera of mice with recent infection. Band of 39kDa was a good marker for the infection; reacting with both low-avidity IgG in recent infection and with high-avidity IgG in latent one. Bands of 15, 23, 30, 60, 66, and 97kDa reacted with variable avidity in both phases of infection. Two antigenic bands were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the GIPLs-enriched fraction of tachyzoite, the 14- and 30-kDa band. The 14-kDa band was recognized by IgM in pooled serum samples of recently infected mice only, while the 30-kDa band was recognized by serum samples of both recent and latent phases of infections. The study highlights the value of avidity immunoblotting assay to discriminate between recent and latent experimental toxoplasmosis. Further study must be carried on human to evaluate the values of the used technique.

Stabilization of oligomeric structure of α‐crystallin by Zn+2 through intersubunit bridging

α-Crystallin, the major protein of mammalian eye lens, is a member of the small heat shock protein family and is a molecular chaperone. We previously reported that its molecular chaperone function as well as stability increased in presence of Zn+². Despite the effect of Zn+² on the structure and function of α-crystallin, evidence for direct interaction between them remained elusive. We now present the MALDI mass spectrometric data that shows direct evidence of Zn+² binding to recombinant αA- and αB-crystallin. The binding stoichiometry was over three Zn+² per subunit of α-crystallin at zinc/protein molar ratio of 20. Observation of multiple Zn+² binding is consistent with the large increase in thermodynamic stability. Sequence-based analysis of αA- and αB-crystallin predicted both proteins to be nonzinc binding proteins. Our dynamic light scattering data shows that Zn+² stabilizes the oligomeric structure of α-crystallin by bridging neighboring subunits in multiple centers. Despite the low affinity binding, the intersubunit bridging by multiple Zn+² makes the oligomer so stable that oligomer breakdown does not occur even at 6M urea. The subunit bridging has been supported by our FRET data that showed absence of subunit exchange in presence of zinc. MALDI data also showed that the interaction of α-crystallin with Zn+² is quite different from other bivalent metal ions. Bound Zn+² could be easily removed by dialysis of the complex. The relevance of such weak interaction on the stability of the oligomeric structure of α-crystallin and its function in the eye lens has been discussed.

Potentiometric urea biosensor based on multi-walled carbon nanotubes (MWCNTs)/silica composite material

A novel potentiometric urea biosensor has been fabricated with urease (Urs) immobilized multi-walled carbon nanotubes (MWCNTs) embedded in silica matrix deposited on the surface of indium tin oxide (ITO) coated glass plate. The enzyme Urs was covalently linked with the exposed free –COOH groups of functionalized MWCNTs (F-MWCNTs), which are subsequently incorporated within the silica matrix by sol–gel method. The Urs/MWCNTs/SiO2/ITO composite modified electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, thermal gravimetric analysis (TGA) and UV–visible spectroscopy. The morphologies and electrochemical performance of the modified Urs/MWCNTs/SiO2/ITO electrode have been investigated by scanning electron microscopy (SEM) and potentiometric method, respectively. The synergistic effect of silica matrix, F-MWCNTs and biocompatibility of Urs/MWCNTs/SiO2 made the biosensor to have the excellent electro catalytic activity and high stability. The resulting biosensor exhibits a good response performance to urea detection with a wide linear range from 2.18×10−5 to 1.07×10−3M urea. The biosensor shows a short response time of 10–25s and a high sensitivity of 23mV/decade/cm2.

Mechanisms of amphipathic helical peptide denaturation by guanidinium chloride and urea: a molecular dynamics simulation study

Urea and GdmCl are widely used to denature proteins at high concentrations. Here, we used MD simulations to study the denaturation mechanisms of helical peptide in different concentrations of GdmCl and urea. It was found that the helical structure of the peptide in water simulation is disappeared after 5ns while the helicity of the peptide is disappeared after 70ns in 2M urea and 25ns in 1M GdmCl. Surprisingly, this result shows that the helical structure in low concentration of denaturants is remained more with respect to that solvated in water. The present work strongly suggests that urea interact more preferentially to non-polar and aromatic side chains in 2M urea; therefore, hydrophobic residues are in more favorable environment in 2M urea. Our results also reveal that the hydrogen bonds between urea and the backbone is the dominant mechanism by which the peptide is destabilized in high concentration of urea. In 1M and 2M GdmCl, GdmCl molecules tend to engage in transient stacking interactions with aromatics and hydrophobic planar side chains that lead to displacement of water from the hydration surface, providing more favorable environment for them. This shows that accumulation of GdmCl around hydrophobic surfaces in 1M and 2M GdmCl solutions prevents proper solvation of the peptide at the beginning. In high GdmCl concentrations, water solvate the peptide better than 1M and 2M GdmCl. Therefore, our results strongly suggest that hydrogen bonds between water and the peptide are important factors in the destabilization of peptide in GdmCl solutions.

Combination of Epstein–Barr virus scaffold (BdRF1/VCA-p40) and small capsid protein (BFRF3/VCA-p18) into a single molecule for improved serodiagnosis of acute and malignant EBV-driven disease

Current single Epstein–Barr virus (EBV) markers fail to reach 100% sensitivity for serodiagnosis of acute and malignant diseases associated with EBV infection. Previous study had identified immunodominant epitopes of VCA-p40 and VCA-p18, and indicated that these two VCA antigens may have diagnostic value for EBV-related diseases. A recombinant protein of the full-length BdRF1 fused to the immunodominant domain of BFRF3 as 6-his tagged protein in Escherichia coli was developed. The recombinant protein was extracted in 8M urea solution and purified by metal-affinity chromatography yielding a 55kDa product (VCA-p40+18). VCA-p40+18 blot-strips examined for IgM reactivity in infectious mononucleosis samples yielded 100% sensitivity and specificity, with improved reactivity compared with IgM/VCA-p18-ELISAs.A recent study described a synthetic peptide-based IgA/[EBNA1+VCA-p18]-ELISA (IgA/EBV-ELISA), with a sensitivity of 90% for diagnosing nasopharyngeal carcinoma. Immunoblot analysis of biopsy-confirmed nasopharyngeal carcinoma cases with low or negative IgA/EBV-ELISA showed 100% IgG reactivity to VCA-p40 and VCA-p18 proteins. Evaluation of VCA-p40+18 as an additional marker for screening and diagnosis of nasopharyngeal carcinoma was carried out. The data showed positive IgA/VCA-p40+18 reactivity by ELISA for 63.6% (14 of 22) nasopharyngeal carcinoma samples that were missed by peptide-based IgA/EBV-ELISA, suggested VCA-p40+18 as an improved marker for nasopharyngeal carcinoma serodiagnosis. The VCA-p40+18 may be combined with an EBNA1 synthetic peptide as an antigen mixture in one or separate IgA ELISA for improved nasopharyngeal carcinoma serodiagnosis.

Solid state fermentation and characterization of partially purified thermostable mannanase from Bacillus sp. MG-33

Bacillus sp. MG-33 was isolated from the desert of Rajasthan (India). The organism produced 500 and 200 Ug-1 of thermostable mannanase (after 96h) in solid state fermentation (SSF) of wheat bran and wheat straw rich-soda pulp at the moisture ratio of 1:1.5 and 1:3 at 30ºC, respectively. Two-step partially purified mannanase was optimally active at 65ºC and was 100% thermostable at 55 to 60ºC for 2h and also retained more than 50% residual activity at 65ºC for 2h. A pH of 6.5 was optimum for enzyme activity and 100% stability up to 4h at this pH. Mannanase activity was slightly enhanced by Ca2+, Fe3+, and Mg2+, while 100% activity was retained in the presence of Ba3+, Li+, and NiCl2 at 1.0-10mM. 1M NaCl and urea did not reduce the enzyme activity. The Km and Vmax of mannanase were 0.2mgml-1 and 60Umg-1ml-1, respectively. Hydrolysis of locust bean was rapid and linear between 5 and 20 min, and ~300µgml-1 mannose was obtained after 20 min of catalytic reaction by enzyme at 65ºC. TLC was used to confirm the mannose as an end product after hydrolysis of locust bean gum.

Proteomic analysis of drug metabolizing networks in renal cell carcinomas with differential drug sensitivity and VHL expression.

e13137 Background: Kidney tumors with mutated VHL constitute 75% of renal cancers. The VHL activity dependent therapeutic outcome is the current focus of translational research in renal oncology. We observed that CAKI-2 kidney cancer cells with wt VHL are more sensitive to sorafenib than 786-O cells with mt VHL, which are more sensitive to temsirolimus (Singhal SS et al 2009). Hence, we performed proteomic analysis to investigate the molecular basis of differential drug sensitivity. Methods: The cell cultures were performed in RPMI medium at 37°C in 5% CO2. The cells were lysed in buffer containing 20mM Tris, 50mM Nacl, 6M urea, 10mM NaPP, 1mM NaF, and 1mM Na3VO4. The lysate (200μg of protein) was subjected to reduction and alkylation of cysteins using 2.5mM DTT and 7mM idoacetamide followed by trypsin digestion and solid phase extraction using a C-18 cartridge (Waters, USA). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed on a linear ion trap–Fourier transform ion cyclotron resonance tandem mass spectrometer (LTQ-FT, Thermo) coupled to an Eksigent nano-LC system. MS/MS spectra were searched against a human protein database by the Mascot software (Matrix Science) and Label-free quantification was done by using spectral counts from the Scaffold program (Proteome Software) with previously validated method (Prokai et al 2009). Results: We detected significant differences in the levels of phase I and II drug metabolizing enzymes in two cell types (Table). Carbonyl reduction catalyzed by AKR1C1 and UGDH involved glucorunidation are vital both in the activation and inactivation of drugs. NQO1 mediates the anticancer effects of beta lapachone (Bey et al. 2006). Conclusions: Our proteomic identification of vital differences in the principal enzymes of drug metabolizing networks identify key molecular determinants of drug sensitivity, which may be further investigated and developed as drug sensitivity biomarkers for clinical use. Proteins identified by LC-MS/MS Fold increase in spectra in CAKI-2 cells comparedto 786-O cells Aldo-keto reductase (family 1 member C1, AKRC1) 6.1 NAD(P)H quinone dehydrogenase 1 (NQO1) 2.8 UDP-glucose 6- dehydrogenase (UGHD) 5.2 No significant financial relationships to disclose.

Multiscale investigation of chemical interference in proteins

We developed a multiscale approach (MultiSCAAL) that integrates the potential of mean force obtained from all-atomistic molecular dynamics simulations with a knowledge-based energy function for coarse-grained molecular simulations in better exploring the energy landscape of a small protein under chemical interference such as chemical denaturation. An excessive amount of water molecules in all-atomistic molecular dynamics simulations often negatively impacts the sampling efficiency of some advanced sampling techniques such as the replica exchange method and it makes the investigation of chemical interferences on protein dynamics difficult. Thus, there is a need to develop an effective strategy that focuses on sampling structural changes in protein conformations rather than solvent molecule fluctuations. In this work, we address this issue by devising a multiscale simulation scheme (MultiSCAAL) that bridges the gap between all-atomistic molecular dynamics simulation and coarse-grained molecular simulation. The two key features of this scheme are the Boltzmann inversion and a protein atomistic reconstruction method we previously developed (SCAAL). Using MultiSCAAL, we were able to enhance the sampling efficiency of proteins solvated by explicit water molecules. Our method has been tested on the folding energy landscape of a small protein Trp-cage with explicit solvent under 8M urea using both the all-atomistic replica exchange molecular dynamics and MultiSCAAL. We compared computational analyses on ensemble conformations of Trp-cage with its available experimental NOE distances. The analysis demonstrated that conformations explored by MultiSCAAL better agree with the ones probed in the experiments because it can effectively capture the changes in side-chain orientations that can flip out of the hydrophobic pocket in the presence of urea and water molecules. In this regard, MultiSCAAL is a promising and effective sampling scheme for investigating chemical interference which presents a great challenge when modeling protein interactions in vivo.

Purification and characterization of an Anti-proliferative and mitogenic plant lectin from tubers of Arisaema speciosum

A novel lectin having specificity for N-acetyllactosamine was purified from the tubers of Arisaema speciosum Martius (family: Araceae) by affinity chromatography on asialofetuin-linked amino activated silica beads. The lectin have four identical subunits of 13.5 kDa each and native molecular mass of 51.2 kDa. ASL agglutinated rabbit, rat, sheep and guinea pig erythrocytes but human ABO blood group erythrocytes were not even after neuraminidase treatment. ASL gave as single band on native PAGE, pH 4.5 and multiple bands in isoelectric focusing and at PAGE 8.3. ASL is a glycoprotein with 0.7% carbohydrate content, stable up to 50°C and at pH 2 to 10. The lectin had no requirement for divalent cations and was stable up to 4M Urea, 3M thiourea and 3M Guanidine-HCl. ASL was rich in acidic amino acids but cysteine was completely absent. Amino acid modification revealed the involvement of tryptophan and tyrosine residues in lectin sugar interaction. The secondary structure estimated in far UV CD spectrum was 38% α-helix, 27% β-sheet and 35% random contributions. ASL showed mitogenic potential towards human peripheral blood mononuclear cell. ASL produced more that 50% growth inhibition of seven human cancer cell lines viz. SW-620, IMR-32, PC-3, SK-N-SH, Colo-205, HCT-15 and HT-29 at 100 μg/ml out of eleven cell lines used.

Backbone additivity in the transfer model of protein solvation

The transfer model implying additivity of the peptide backbone free energy of transfer is computationally tested. Molecular dynamics simulations are used to determine the extent of change in transfer free energy (DeltaG(tr)) with increase in chain length of oligoglycine with capped end groups. Solvation free energies of oligoglycine models of varying lengths in pure water and in the osmolyte solutions, 2M urea and 2M trimethylamine N-oxide (TMAO), were calculated from simulations of all atom models, and DeltaG(tr) values for peptide backbone transfer from water to the osmolyte solutions were determined. The results show that the transfer free energies change linearly with increasing chain length, demonstrating the principle of additivity, and provide values in reasonable agreement with experiment. The peptide backbone transfer free energy contributions arise from van der Waals interactions in the case of transfer to urea, but from electrostatics on transfer to TMAO solution. The simulations used here allow for the calculation of the solvation and transfer free energy of longer oligoglycine models to be evaluated than is currently possible through experiment. The peptide backbone unit computed transfer free energy of -54 cal/mol/M compares quite favorably with -43 cal/mol/M determined experimentally.

Approaches to efficient production of recombinant angiogenesis inhibitor rhVEGI‐192 and characterization of its structure and antiangiogenic function

Methods to prepare pure, bioactive recombinant human vascular endothelial growth inhibitor (rhVEGI), a potent inhibitor of angiogenesis potentially applicable in antiangiogenic cancer therapy, are in urgent demand for preclinical investigation as well as future clinical trials of the protein. Here, we report expression and purification of rhVEGI-192, a recombinant VEGI isoform, comparatively using host strains BL21 (DE3) pLysS and Origami B (DE3) with IPTG-induction and autoinduction techniques. Our study identified that a combined use of Origami B (DE3) strain and autoinduction expression system gave rise to a high yield of purified rhVEGI-192 at 105.38 mg/L culture by immobilized-metal affinity chromatography on Ni-NTA column. The antiangiogenic activity was effectively restored after the insoluble fractions being dissolved in 8M urea and subsequently subjected to a gradient-dialysis refolding process. Functional tests demonstrated that the purified rhVEGI-192 potently inhibited endothelial growth, induced endothelial apoptosis and suppressed neovascularization in chicken chorioallantoic membrane, indicating that the developed method allows preparation of rhVEGI-192 with high yield, solubility, and bioactivity. Most importantly, our study also demonstrates that VEGI-192 is capable of forming polymeric structure, which is possibly required for its antiangiogenic activity.

Backbone and stereospecific beta-sidechain assignments of 1H, 13C and 15N for Ubiquitin Unfolded in 8M Urea, pH2.5.

Backbone and stereospecific beta-sidechain assignments of 1H, 13C and 15N for Ubiquitin Unfolded in 8M Urea, pH2.5.

Jk(a-b-) phenotype screening by the urea lysis test in Thai blood donors.

The Jk(a-b-) phenotype is rare in most populations and often detected after transfusion or pregnancy. After immunisation, anti-Jk3 forms and it can be difficult to find compatible Jk(a-b-) donors. Using anti-Jk(a) and anti-Jk(b) in a conventional tube method is unsuitable for identifying Jk(a-b-) in mass screening of blood donors. Jk(a-b-) phenotypes are associated with the absence of urea transporters on erythrocytes, making red blood cells (RBC) resistant to lysis by 2M urea, while Jk(a+b-), Jk(a-b+) and Jk(a+b+) phenotypes are susceptible to lysis. We screened for Jk(a-b-) phenotypes in blood donors by the urea lysis test using a 96-well microplate. The Jk(a-b-) phenotypes were confirmed by the indirect antiglobulin test (IAT). Altogether, 20,163 blood samples from Thai blood donors were tested and only RBC from five samples were resistant to lysis by 2M urea, while 20,158 samples were completely lysed within 5 min. In an IAT, both anti-Jk(a) and anti-Jk(b) failed to agglutinate RBC from all five samples. Using a micro-titre plate, the direct urea lysis test, costs * 0.01, about 480 times less than IAT. Moreover, the test time for each plate (94 samples) is about 18 times less than that for IAT. Jk(a-b-) phenotype screening by the direct urea lysis test on samples in a micro-titre plate is simple, cost-effective and practical for mass screening of blood donors.