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The benefits of Eurycoma longifolia (Tongkat Ali, TA) for its aphrodisiac capabilities are well known and many products are marketed worldwide. Due to its popularity, the plant is being abused for promoting fake products. Therefore, there is a need for better testing of the markers required by authorities and responsible manufacturers. A low-molecular-weight protein has been studied for developing it as a testing marker. Two dimensional electrophoresis (2DE) (four spots were observed) was used for positive detection of proteins in an aqueous extract of TA root and the pronounced separation of a Coomassie-stained spot, subsequently referred to as Marker A. Consecutive chromatographic separations of the aqueous extract of TA led to the isolation of pure protein from Marker A. When this marker was used to test 46 TA-based products randomly selected from markets worldwide, in 20 of them, the results were found to be comparable to those obtained using the organic eurycomanone marker. The ranking of products from the highest quantity to the lowest observed to be ordered differently if compared for both markers. This is an expected outcome because Marker A was measured for its protein content and eurycomanone for its organic molecule. Marker A detection using 2DE is shown to be a useful tool to test products supplemented with E. longifolia root. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was production of a recombinant antigen from envelope protein of dengue virus, in order to achieve high level expression in soluble form. Therefore, the possibility of envelope protein domain III expression in bacterial host was studied. Methods: Multiple sequence alignment for domain III sequences was carried out using Megalign software. The Modeller and Optimizer software were used for protein structure prediction and sequence optimization. After gene synthesis and subcloning in the pET21a expression vector, the optimization of recombinant protein expression was carried out in Escherichia coli. Ni-NTA chromatography columns were used for protein purification. The efficiency of protein expression was analyzed using electrophoresis and western blotting methods. Findings: A consensus sequence for domain III of dengue virus type 2 envelope protein was provided. The structural properties of target protein were predicted using bioinformatics methods. For high expression level, the sequence of EDIII2 gene was optimized for appropriate codon usage and GC content. The coding gene sequence was approved by sequencing and enzymatic digestion. For disulfide bound formation in protein structure, the Origami (DE3) expression host was used. At the final step, expression of target protein was optimized, and a high concentration of recombinant protein was achieved in soluble form. Conclusion: The results of this study showed that the optimization of domain III sequence led to obtain high concentration (20 mg/l) of produced protein. This expression system can be used for production of dengue virus envelope protein domain III. [ABSTRACT FROM AUTHOR]

Purpose. Pigment epithelium-derived factor (PEDF) is a multifunctional serpin. The purpose of this study is to identify PEDF protein forms and investigate their biological activities on tumor cell lines. Methods. Recombinant human PEDF proteins were purified by cation- and anion-exchange column chromatography. They were subjected to SDS-PAGE, IEF, deglycosylation, heparin affinity chromatography, and limited proteolysis. Cell viability, real-time electrical impedance of cells, and wound healing assays were performed using bladder and breast cancer cell lines, rat retinal R28, and human ARPE-19 cells. Results. Two PEDF protein peaks were identified after anion-exchange column chromatography: PEDF-1 eluting with lower ionic strength than PEDF-2. PEDF-1 had higher pI value and lower apparent molecular weight than PEDF-2. Both PEDF forms were glycosylated, bound to heparin, and had identical patterns by limited proteolysis. However, PEDF-2 emerged as being highly potent in lowering cell viability in all tumor cell lines tested, and in inhibiting tumor and ARPE-19 cell migration. In contrast, PEDF-1 minimally affected tumor cell viability and cell migration but protected R28 cells against death caused by serum starvation. Conclusion. Two distinct biochemical forms of PEDF varying in overall charge have distinct biological effects on tumor cell viability and migration. The existence of PEDF forms may explain the multifunctional modality of PEDF. [ABSTRACT FROM AUTHOR]

Acetylation of nascent protein Nα-termini is a common modification among archae and eukaryotes and can influence the structure and function of target proteins. This modification has been studied on an individual protein or (synthetic) peptide level or on a proteome scale using two-dimensional polyacrylamide gel electrophoresis. We recently developed mass spectrometry driven proteome analytical approaches specifically targeting the amino (N) terminus of proteins based on the concept of diagonal reverse-phase chromatography. We here review how this so-called combined fractional diagonal chromatography (COFRADIC) technique can be used in combination with differential mass-tagging strategies as to both qualitatively and quantitatively assess protein Nα-acetylation in whole proteomes. [ABSTRACT FROM AUTHOR]

MS/MS techniques are well customized now for proteomic analysis, even for non-sequenced organisms, since peptide sequences obtained by these methods can be matched with those found in databases from closely related sequenced organisms. We used this approach to characterize the protein content of the “Rovabio™ Excel”, an enzymatic cocktail produced by Penicillium funiculosum that is used as feed additive in animal nutrition. Protein separation by bi-dimensional electrophoresis yielded more than 100 spots, from which 37 proteins were unambiguously assigned from peptide sequences. By one-dimensional SDS-gel electrophoresis, 34 proteins were identified among which 8 were not found in the 2-DE analysis. A third method, termed ‘peptidic shotgun’, which consists in a direct treatment of the cocktail by trypsin followed by separation of the peptides on two-dimensional liquid chromatography, resulted in the identification of two additional proteins not found by the two other methods. Altogether, more than 50 proteins, among which several glycosylhydrolytic, hemicellulolytic and proteolytic enzymes, were identified by combining three separation methods in this enzymatic cocktail. This work confirmed the power of proteome analysis to explore the genome expression of a non-sequenced fungus by taking advantage of sequences from phylogenetically related filamentous fungi and pave the way for further functional analysis of P. funiculosum. [ABSTRACT FROM AUTHOR]

Proteomics has progressed radically in the last 5 years and is now on par with most genomic technologies in throughput and comprehensiveness. Analyzing peptide mixtures by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) has emerged as the main technology for in-depth proteome analysis whereas two-dimensional gel electrophoresis, low-resolution MALDI, and protein arrays are playing niche roles. MS-based proteomics is rapidly becoming quantitative through both label-free and stable isotope labeling technologies. The latest generation of mass spectrometers combines extremely high resolving power, mass accuracy, and very high sequencing speed in routine proteomic applications. Peptide fragmentation is mostly performed in low- resolution but very sensitive and fast linear ion traps. However, alternative fragmentation methods and high-resolution fragment analysis are becoming much more practical. Recent advances in computational proteomics are removing the data analysis bottleneck. Thus, in a few specialized laboratories, "precision proteomics" can now identify and quantify almost all fragmented peptide peaks. Huge challenges and opportunities remain in technology development for proteomics; thus, this is not "the beginning of the end" but surely "the end of the beginning." [ABSTRACT FROM AUTHOR]

The article presents a study which illustrated a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting with intracellular domains of G protein-coupled receptors (GPCR). An introduction to intracellular proteins is offered. Researchers reportedly identified 15 proteins that directly bind or indirectly to the C-terminal tail of the 5-HT2c receptor. Also highlighted is the significance of the developed method.

Abstract: A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase. [Copyright &y& Elsevier]

Previous research on proteins that inhibit kidney stone formation has identified a relatively small number of well-characterized inhibitors. Identification of additional stone inhibitors would increase understanding of the pathogenesis and pathophysiology of nephrolithiasis. We have combined conventional biochemical methods with recent advances in mass spectrometry (MS) to identify a novel calcium oxalate (CaOx) crystal growth inhibitor in normal human urine. Anionic proteins were isolated by DEAE adsorption and separated by HiLoad 16/60 Superdex 75 gel filtration. A fraction with potent inhibitory activity against CaOx crystal growth was isolated and purified by anion exchange chromatography. The protein in 2 subfractions that retained inhibitory activity was identified by matrix-assisted laser desorption/ionization-time-of-flight MS and electrospray ionization-quadrupole-time-of-flight tandem MS as human trefoil factor 1 (TFF1). Western blot analysis confirmed the mass spectrometric protein identification. Functional studies of urinary TFF1 demonstrated that its inhibitory potency was similar to that of nephrocalcin. The inhibitory activity of urinary TFF1 was dose dependent and was inhibited by TFF1 antisera. Anti-C-terminal antibody was particularly effective, consistent with our proposed model in which the 4 C-terminal glutamic residues of TFF1 interact with calcium ions to prevent CaOx crystal growth. Concentrations and relative amounts of TFF1 in the urine of patients with idiopathic CaOx kidney stone were significantly less (2.5-fold for the concentrations and 5- to 22-fold for the relative amounts) than those found in controls. These data indicate that TFF1 is a novel potent CaOx crystal growth inhibitor with a potential pathophysiological role in nephrolithiasis. [ABSTRACT FROM AUTHOR]

The 5-hydroxytryptamine type 4 receptor (5-HT4R) is involved in learning, feeding, respiratory control and gastrointestinal transit. This receptor is one of the G-protein-coupled receptors for which alternative mRNA splicing generates the most variants that differ in their C-terminal extremities. Some 5-HT4R variants (a, e and f) express canonical PDZ ligands at their C-termini. Here, we have examined whether some mouse 5-HT4R variants associate with specific sets of proteins, using a proteomic approach based on peptide-affinity chromatography, two-dimensional electrophoresis and mass spectrometry. We have identified ten proteins that interact specifically with the 5-HT4(a)R and three that only associate with the 5-HT4(e)R. Most of them are PDZ proteins. Among the proteins that associated specifically with the 5-HT4(a)R variant, NHERF greatly modified its subcellular localization. Moreover, NHERF recruited the 5-HT4(a)R to microvilli, where it localized with activated ezrin, consistent with the role of 5-HT4(a)R in cytoskeleton remodelling. The 5-HT4(a)R also interacted with both the constitutive and inducible (upon methamphetamine treatment) forms of the recently cloned sorting nexin 27 (SNX27a and b, respectively). We found that SNX27a redirected part of 5-HT4(a)R to early endosomes. The interaction of the 5-HT4R splice variants with distinct sets of PDZ proteins might specify their cellular localization as well as their signal transduction properties. [ABSTRACT FROM AUTHOR]

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-aibumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65°C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65°C (D65°C values; times required to reduce the concentration of bacteria by a factor of 10 at 65°C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D65°C value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance. [ABSTRACT FROM AUTHOR]

A pregastric lipase was purified from calf pharyngeal tissues. The purification procedure was based on chromatographies on octyl-Sepharose and lentil-lectin-Sepharose followed by gel filtration. The final preparation, with an overall recovery of 26% of activity, gave a single protein band on dodecyl sulfate/polyacrylamide gel electrophoresis with a Mr of 55 000. The Mr on gel filtration was 44–48 000. The discrepancy may be due to the fact that pregastric lipase is a glycoprotein containing &assymp;10% (w/w) of carbohydrate. The pI was around 7.0 and the enzyme protein is characterized by a high content of branched, aliphatic amino acid residues. The NH2-terminal amino acid sequence is: H2N-Phe-Leu/(Ile)-Gly-. Rabbit antibodies to the purified preparation detected only one component in the crude starting material in immuno-blotting experiments. Preincubation with antiserum resulted in loss of enzyme activity, showing that the antibodies were directed against the lipase. [ABSTRACT FROM AUTHOR]

1. CM-cellulose chromatography of a fraction soluble in 5% perchloric acid from Ceratitis capitata chromatin yields three proteins, C1a1, C1a2 and C1b, which have been purified to electrophoretical homogeneity. 2. C1a1, C1a2 and C1b analyse like high mobility group (HMG) non-histone chromosomal proteins, although they do not exactly correspond with those from vertebrates. It is proposed that C1 proteins, as well as Drosophila D1 [Rodriguez Alfageme et al. (1980) Chromosoma, 78, 1-31] are representative of a class of insect-specific HMG proteins. Tryptic fingerprints show that C1a1 and C1a2 are very similar, but C1b is a somewhat distinct protein. Circular dichroism studies have shown that these preparations do non appreciably fold on increasing ionic strength. 3. The interactions between DNA and C1 proteins have been studied. These proteins precipitate DNA in 0.15 M NaCl, 0.015 M sodium citrate and the precipitation curves are cooperative. Soluble complexes between C1 proteins and DNA could be prepared in low ionic strength media and their thermal denaturation profiles obtained. C1 proteins do not destabilize DNA under the conditions used to prepare the complexes but the three proteins stabilize DNA to a different degree. From these studies it has been concluded that the association constant of C1b to DNA is smaller than that of C1a1 and C1a2. [ABSTRACT FROM AUTHOR]

1. High-voltage electrophoresis and chromatography before and after reaction with 5-dimethylaminonapthalene-1-sulphonyl chloride have identified putrescine and spermidine in hydrolysates of cyst coat proteins from the protozoan Colpoda steinii. 2. Amounts present varied with putrescine up to 19.7 and spermidine up to 16.9 residues per 1000 amino acid residues. 3. The amines were not, in the main, removed by acid or alkaline extraction or by reprecipitation. They were present in hydrolysates of peptides isolated electrophoretically from acid-degraded coat protein. 4. Proteolysis of oxidized coat protein produced a soluble core polypeptide to which the major proportion of the amines were attached and which had a simple composition. It was composed almost entirely of glutamic acid or glutamine, glycine, serine and cysteic acid, these residues being present in the approximate ratio of 10:2:1:1. 5. When coat protein was treated with 5-dimethylaminoaphthalene-1-sulphonyl chloride and hydrolysed no fully substituted amines could be detected but putrescine with one group substituted and spermidine derivatives with one and two groups substituted were present. [ABSTRACT FROM AUTHOR]

Reaction of the thiol reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (Nbs2) with the brain-specific protein S-100 favours stabilization of the quaternary structure of the protein via disulfide bond formation. This process is modulated by those cations (Ca2+ and K2+) which are known to affect the conformation of the protein. Ca2+ markedly favours the reaction of S-100 with Nbs2 but inhibits subsequent disulfide bond formation: K . on the contrary. is much less effective in promoting interaction with Nbs2 but strongly stimulates disulfide bond formation. These findings are inter- preted assuming that in presence of Ca2+ the three subunits forming the native S-100 protein have two cysteine residues exposed to the solvent but mismatched to form disulfides while in presence of Kt tile sulphydrvl groups are in a less accessible position to Nbs2 but suitable for S-S bond formation. C'rosslinkrng of S-100 subunits is characterized by the appearance in dodecylsulphate electrophoresis of two very close protein bands having a molecular weight almost identical to that of the native, undenatured protein but not of higher or lower-molecular-weight components. This finding, and the demonstration that both the crosslinked and native S-100 proteins have identical profiles when analyzed by sucrose density centrifugation or gel chromatography indicate that disulfide bond formation occurs among subunits of the same molecule. [ABSTRACT FROM AUTHOR]

Tryptophan synthase was purified from Saccharomyces cerevisiae with the aid of affinity chromatography up to a specific activity of 525 units/g protein. Electrophoresis on polyacrylamide gel showed one main protein component. Gel filtration on Sephadex G-150 indicates an average molecluar weight of 143 000. Dissociation with dodecylsulfate followed by dodecylsulfate-acrylamide gel electrophoresis leads to the conclusion that the enzyme is probably composed of four subunits of equal size. The Km-values with respect to its substrates indoleglycerol phosphate, indole and L-serine were estimated. L-Serine was shown to increase the Km-value for indoleglycerol phosphate about 7-fold. Several indole analogues inhibit the tryptophan synthase reaction (B) (i.e. indole + serine → tryptophan); indoleacrylic acid is the most effective. The pH-optimum of tryptophan synthase is around 6.9 to 7.0. Sulfhydryl modification by 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuriphenylsulfonic acid results in a large loss of tryptophan synthase activity, which cannot be prevented by the substrates L-serine and indole. The tryptophan synthase inactivating system from yeast affects the catalysis of both reactions (A) (i.e. indoleglycerol phosphate → indole + glyceraldehyde 3-phosphate) and (B).

1. A two-dimensional gel electrophoresis procedure employing a combination of isoelectric focusing and dodecylsulfate elcctrophoresis has been used to analyse the components of 32P-labelled nonhistone protein fractions obtained by the chromatography of salt-urea dissociated chromatins on hydroxyapatite. 2. In this way the nonhistone proteins eluted from hydroxyapatite by 0.05 M phosphate (fraction H2) have been found to consist of a heterogeneous mixture of components of a molecular weight range of 15000 to 200000 and to have isoelcctric points between pH 4.5 and 9. The components of the fraction eluted from hydroxyapatite by 0.2 M phosphate (fraction H3) appeared to consist of a smaller group of proteins with a molecular weight range similar to that of the H2 proteins but whose isoelectric points lay between pH 2 and 6. Both fractions consisted of a mixture of phoshorylated and nonphosphorylated protein species. 3. Comparison of the components of H2 and H3 protein fractions by this electrophoretic procedure showed that many of the phosphorylated and nonphosphorylated proteins were common to mouse liver, kidney and brain chromatins. Only a few species were found to be tissue specific. 4. It was found that chromatography on QAE-Sephadex did not follow the same separation pattern as that obtained by isoelectric focusing, suggesting that the fractionation on this ion- exchange material is not entirely dependent on the overall charge of the polypeptide. 5. Preparation of individual nonhistone protein species has been attempted using this chromatographic procedure in conjunction with gel filtration in guanidine hydrochloride. [ABSTRACT FROM AUTHOR]

A new papain inhibitor was purified from psoriatic epidermal scales using gel chromatography and anion exchange chromatography. The purified protein inhibited papain and ficin but not cathepsin B, cathepsin H, trypsin, or chymotrypsin. Isoelectric focusing revealed 3 major inhibitor variants with pI's of 7.3, 6.9, and 6.5. A Mr of 38,000 was obtained by a gel chromatographic method for the crude inhibitor. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr values of the isoelectric variants were: 43,000 for the variant pI 7.3, 43,000 and 35,000 for the variant pI 6.9, and 34,000-35,000 for the variant pI 6.5. An antiserum of the inhibitor was used to locate the inhibitor in the psoriatic and normal epidermis. In psoriatic epidermis, the inhibitor was found in the peripheral cytoplasm of spinous cells and in the scale. In normal epidermis, the staining was seen only in orifices of hair follicles. An inhibitor with similar size and antigenic properties to that isolated from the psoriatic scales was demonstrated in extracts made from the whole-thickness epidermis but not in extracts from the healthy epidermal scales, the dermis, the liver, the spleen, or the blood serum. [ABSTRACT FROM AUTHOR]

The interaction of FaeE, a periplasmic chaperone involved in K8B biosynthesis, and the major fimbrial subunit FaeG was investigated. The genes encoding the two proteins were subcloned together in the expression vector pINIIIAL Cells expressing the subcloned genes accumulated in their periplasm a complex of FaeE and FaeG. This complex was purified by isoelectric focusing and anion-exchange fast-protein liquid chromatography. SDS-PAGE, native gel electrophoresis, immunoblotting and determination of the N-terminal amino acid sequences and the molar ratio of the N-terminal amino acid residues revealed that the complex is a heterotrimer consisting of two molecules of FaeE and one molecule of FaeG. The periplasmic chaperone FaeE was purified from the periplasm of cells expressing only the subcloned faeE gene. Gel filtration, protein cross-linking analysis and a biophysical approach in which the rotation diffusion coefficient of the purified FaeE was determined led to the conclusion that the native FaeE chaperone is a homodimer. [ABSTRACT FROM AUTHOR]