Search

We have developed methods for producing recombinant proteins of Noggin family (Noggin1 and Noggin2 of the Xenopus laevis frog) that can interact with BMP factors of TGF-beta superfamily. The genetic constructs which allow one to effectively obtain Noggin1 and Noggin2 from synthetic mRNA microinjected into Xenopus laevis early embryos, as well as in the prokaryotic expression system, were generated. The obtained proteins contain three Myc-tag epitopes on their N-terminus. This allow one to compare the expression levels of Noggin1 and Noggin 2 constructs, to purify them on the affine immunosorbent and to show the activity of Noggin proteins by analyzing their ability to bind BMP4 factor TGF-beta surperfamily by co-immunoprecipitation.

As a highly efficient and easily absorbable source of chromium, the identities of the chromium-binding substances in yeast remain unclear. In this study, a mild extraction procedure involving extraction with ammonia, three-gel filtration, and high-performance liquid chromatography was adopted to obtain two chromium-binding substances from high-chromium yeast. A low-molecular-weight chromium-binding substance was identified, with mass-to-charge ratios (m/z) of 769 and 712, which included glutamic acid, glycine, and cysteine in an approximate ratio of 1:1:1, as well as nicotinic acid and chromium(III). Furthermore, it significantly potentiated (by 51%) the action of insulin to stimulate the conversion of (14)C-glucose into lipid in adipocytes. A novel high-molecular-weight chromium-binding substance was also isolated: electrospray ionization tandem mass spectrometry tentatively identified it as HUB1 target protein-1, glyceraldehyde-3-phosphate dehydrogenase, or ribosomal protein L2A(L5A)(rp8)(YL6). This is the first report of a high-molecular-weight chromium-binding substance in yeast and merits further studies.

Numb is an endocytic adaptor protein that regulates the endocytosis and trafficking of transmembrane receptors including Notch, E-cadherin, and integrins. Vertebrate Numb is alternatively spliced at exons 3 and 9 to give rise to four protein isoforms. Expression of these isoforms varies at different developmental stages, and although the function of Numb isoforms containing exon 3 has been studied, the role of exon 9 inclusion has not been shown. Here we use affinity purification and tandem mass spectrometry to identify Numb associated proteins, including novel interactions with REPS1, BMP2K, and BCR. In vitro binding measurements indicated exon 9-independent Numb interaction with REPS1 and Eps15 EH domains. Selected reaction monitoring mass spectrometry was used to quantitatively compare the proteins associated with the p72 and p66 Numb isoforms, which differ by the exon 9 region. This showed that significantly more EPS15 and three AP-2 subunit proteins bound Numb isoforms containing exon 9. The EPS15 preference for exon 9-containing Numb was confirmed in intact cells by using a proximity ligation assay. Finally, we used multiplexed selected reaction monitoring mass spectrometry to assess the dynamic regulation of Numb association with endocytic proteins. Numb hyper-phosphorylation resulted in disassociation of Numb endocytic complexes, while inhibition of endocytosis did not alter Numb association with the AP-2 complex but altered recruitment of EPS15, REPS1, and BMP2K. Hence, quantitative mass spectrometric analysis of Numb protein-protein interactions has provided new insights into the assembly and regulation of protein complexes important in development and cancer.

Veterinary antibiotics entering agricultural land with manure pose the risk of spreading antibiotic resistance. The fate of sulfadiazine (SDZ) introduced via manure and its effect on resistance gene levels in the rhizosphere were compared with that in bulk soil. Maize plants were grown for 9 weeks in soil fertilized with manure either from SDZ-treated pigs (SDZ treatment) or from untreated pigs (control). CaCl(2) -extractable concentrations of SDZ dissipated faster in the rhizosphere than in bulk soil, but SDZ remained detectable over the whole time. For bulk soil, the abundance of sul1 and sul2 relative to 16S rRNA gene copies was higher in the SDZ treatment than in the control, as revealed by quantitative PCR on days 14 and 63. In the rhizosphere, sampled on day 63, the relative sul gene abundances were also significantly increased in the SDZ treatment. The accumulated SDZ exposure (until day 63) of the bacteria significantly correlated with the log relative abundance of sul1 and sul2, so that these resistance genes were less abundant in the rhizosphere than in bulk soil. Plasmids conferring SDZ resistance, which were exogenously captured in Escherichia coli, mainly belonged to the LowGC group and carried a heterogeneous load of resistances to different classes of antibiotics., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Midkine is a heparin-binding growth factor that promotes cell growth, survival, and migration. Externally added midkine prevents ventricular remodeling and improves long-term survival after myocardial infarction in the mouse. Preclinical testing of this protein is in progress. Externally added pleiotrophin, a member of the midkine protein family, promotes functional recovery after neural transplantation in rats. Thus, pleiotrophin is also a candidate therapeutic protein. Large amounts of these proteins were obtained by using the heterologous protein expression system of Pichia pastoris, and the recombinant P. pastoris clones were cultured in a controlled fermentor. Intracellular expression yielded about 300 mg/L recombinant human (rh)-midkine, which was extracted, renatured, and purified. From 1 L of the culture, 64 mg of rh-midkine was purified. Secretory expression induced by the midkine secretion signal resulted in about 100 mg of rhmidkine in 1 L of the culture supernatant, but over 70% of the rh-midkine had yeast-specific glycosylation. Three threonyl residues that are targets for glycosylation were substituted with alanyl residues, and nonglycosylated, active rh-midkine was obtained. In secretory expression using α-mating factor prepro-sequence, about 640 mg/L rh-midkine was obtained, but it was partially truncated. Therefore, a protease-deficient host was used, and about 360 mg/L intact rh-midkine was then obtained. The rh-midkine was recovered and purified, with 70% final yield. All purified rh-midkine, regardless of expression method, was able to promote mammalian cell proliferation. In secretory expression of rh-pleiotrophin using α- mating factor prepro-sequence, 260 mg/L rh-pleiotrophin could be secreted. The rh-pleiotrophin was recovered and efficiently purified with 72% final yield.

Cilia, the hair-like protrusions found on most eukaryotic cells, were once considered vestigial organelles. The recent renaissance of research in cilia arose from the discoveries of intraflagellar transport (IFT) and the involvement of IFT particle proteins in human diseases. Many IFT particle proteins have since been identified, and research on IFT particle complexes and their protein components continues to provide insight into the mechanism of IFT and the etiology of ciliopathies. In this chapter, we describe the methods of isolating IFT particles from the flagella of Chlamydomonas reinhardtii. Two methods, sucrose density gradient fractionation and immunoprecipitation, are explained in detail. Troubleshooting information is presented to illustrate the critical steps of the procedure to ensure successful implementation of these methods in individual labs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Angiogenesis, a formation of neovessels, is regulated by the local balance between angiogenesis stimulators and inhibitors. A number of such endogenous regulators of angiogenesis have been found in the body. Recently, vasohibin-1 (VASH1) was isolated as a negative feedback regulator of angiogenesis produced by endothelial cells (ECs) and subsequently vasohibin-2 (VASH2) as a homologue of VASH1. It was then explored that VASH1 is expressed in ECs to terminate angiogenesis, whereas VASH2 is expressed in cells other than ECs to promote angiogenesis in the mouse model of angiogenesis. This review will focus on the vasohibin family members, which are novel regulators of angiogenesis.

Steroidogenic acute regulatory-related lipid transfer (START) domain proteins are involved in the nonvesicular intracellular transport of lipids and sterols. The STARD1 (STARD1 and STARD3) and STARD4 subfamilies (STARD4-6) have an internal cavity large enough to accommodate sterols. To provide a deeper understanding on the structural biology of this domain, the binding of sterols to STARD5, a member of the STARD4 subfamily, was monitored. The SAR by NMR [(1)H-(15)N heteronuclear single-quantum coherence (HSQC)] approach, complemented by circular dichroism (CD) and isothermal titration calorimetry (ITC), was used. Titration of STARD5 with cholic (CA) and chenodeoxycholic acid (CDCA), ligands of the farnesoid X receptor (FXR), leads to drastic perturbation of the (1)H-(15)N HSQC spectra and the identification of the residues in contact with those ligands. The most perturbed residues in presence of ligands are lining the internal cavity of the protein. Ka values of 1.8·10-(4) M(-1) and 6.3·10(4) M(-1) were measured for CA and CDCA, respectively. This is the first report of a START domain protein in complex with a sterol ligand. Our original findings indicate that STARD5 may be involved in the transport of bile acids rather than cholesterol.

Full-length cDNA for the pig metallothionein 1A (pMT1A) gene was synthesized based on the pig MT1A gene sequence in Genbank and cloned into the pMD18-T vector. After sequence analysis and structure prediction, the pMT1A gene was cloned into vector pET-32a (+) containing a His-tag. The recombinant pMT1A (rpMT1A) was expressed in a soluble form using Escherichia coli Rosetta™ (DE3) plysS cells. Western blotting showed that the purified rpMT1A protein bound an anti-His-tag monoclonal antibody. Further investigation revealed that the rpMT1A protein showed high metal-binding activity with the divalent metal ions copper (Cu²⁺), zinc (Zn²⁺), and cadmium (Cd²⁺).

Under phosphate starvation conditions, Escherichia coli can utilize sn-glycerol-3-phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68 ± 0.02 μM and 5.1 ± 0.3 μM, respectively, while glycerol-2-phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8 Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp-169 for G3P binding. The purified UgpAEC2 complex displayed UgpB/G3P-stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG-UgpC could be functionally reconstituted while a UgpAE-MalK complex was unstable., (© 2012 Blackwell Publishing Ltd.)

Plant nonspecific lipid transfer proteins are small basic proteins that transport phospholipids between membranes and are subdivided into two subfamilies, nsLTP(1) (9 kDa) and nsLTP(2) (7 kDa). LTPs have potential application in the defense reactions against pathogens and the drug delivery systems. Many efforts have been made for purification of different nsLTPs from various plants; however, most of them used successive purification procedures. We have developed a relatively simple and efficient method for the purification of rice nsLTP(1), based on the proteolytic activity of kiwifruit actinidin on the rice seed extract and one-step chromatographic procedure on a CM-Sepharose column. The purity of protein was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The isolated LTP(1) migrated as a homogenous polypeptide with molecular mass of 9 kDa that confirms the efficiency of actinidin on the digestion of major contaminations present in the rice seed extract without any harmful effect on the LTP(1). The advantages of using proteolytic activity of actinidin in purifying rice LTP(1) includes the reduced separation time allowing the purification of LTP(1) in one-step chromatographic procedure, low costing, high efficiency, and the relative simplicity of the method., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cell penetrating peptides (CPPs) belong to the large family of membrane active peptides that comprises antimicrobial and viral fusion peptides with whom they share many properties. CPPs have been increasingly used to transport a wide range of molecules and nanoparticles inside cells. Despite their recognized potential transporting properties, their mode of action is far from being understood and has been a matter of debate. Penetratin, a widely used CPP is one of the first discovered CPPs, yet its mechanism of action remains obscure. Herein an overview on studies regarding cellular and liposomal uptake and the interaction with lipid model systems of CPPs and more particularly penetratin is provided. Special emphasis will be given to biophysical approaches to investigate penetratin/lipid interaction and subsequent lipid reorganization using lipid model systems.

The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Midkine (MDK) and Pleiotrophin (PTN) belong to a class of heparin-binding growth factors and are highly expressed in a number of cancers. Bioactive and recombinant MDK and PTN are critical reagent for cancer drug discovery studies. MDK and PTN belong to a newly evolving family of secreted neurotrophic and developmentally regulated heparin-binding molecules. PTN is related to MDK with 45% sequence identity and both proteins have been shown to be involved in promoting neurite outgrowth. MDK is a cysteine-rich 13kDa protein containing five disulfide bonds and PTN is 19kDa protein containing ten disulphide bonds. In this study, we expressed recombinant human MDK (rhMDK), mouse MDK (rmMDK) and human pleiotrophin (rhPTN) in Escherichia coli BL21(DE3)pLysS strain. Soluble rhMDK, rmMDK and rhPTN were expressed at a high-level in this strain and the protein was purified (∼90%) by a one-step purification using heparin affinity chromatography. A total of 4mg purified MDK and 7mg of purified PTN were obtained with the overall yield from 1L of bacterial culture. Activity of purified rhMDK and rhPTN was confirmed by a cell proliferation assay using NIH3T3 cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Prolactin inducible protein (PIP) is a ~17 kDa protein, which is known to play vital roles in immunoregulation, fertility, antimicrobial activity, apoptosis and tumour progression., Objectives: This study reports quantification of PIP concentration in human seminal plasma (SP) samples., Methodology: PIP was purified by immunoprecipitation and its concentration in human SP samples was quantified by ELISA method., Results: Average concentration of PIP in normozoospermia, oligozoospermia and azoospermia was 290.3 ± 71.5 µg/mL, 306.4 ± 71.2 µg/mL and 60.5 ± 23.6 µg/mL respectively., Conclusion: There was no significant variation in PIP levels in normozoospermia and oligozoospermia while its expression was down-regulated in azoospermia, indicating that PIP may be a plausible marker of azoospermia.

The human thioredoxin (TRX)-interacting protein is found in multiple subcellular compartments and plays a major role in redox homeostasis, particularly in the context of metabolism (e.g., lipidemia and glycemia) and apoptosis. A molecular approach to the protein's modus operandi is still needed because some aspects of the TRX-interacting protein-mediated regulation of TRX are not clearly understood. To this end, His-tagged TRX-interacting proteins were over-expressed in Escherichia coli. Because the protein is expressed mainly in inclusion bodies, it was denatured in high concentrations of guanidium hydrochloride, centrifuged, and purified by Ni-NTA affinity chromatography. His-TRX-interacting protein was then refolded by dialysis and its restructuring monitored by circular dichroism spectrometry. This preparation resulted in the formation of a covalent complex with recombinant human TRX, demonstrating that association occurs without the intervention of other partner proteins. Multiple cysteine-to-serine mutants of TRX-interacting protein were produced and purified. These mutations were efficient in limiting the formation of disulfide-linked homo-oligomers in an oxidizing environment. The mutants were also used to gain functional insight into the formation of the TRX-interacting protein-TRX complexes. These complexes were able to form in the absence of internal disulfide bridges. A mutant with all but one cysteine changed to serine (Cys ²⁴⁷) also showed an enhanced capacity to form complexes with TRX demonstrating, in a pure molecular system, that this particular cysteine is likely responsible for the disulfide bridge between TRX-interacting protein and TRX., (Copyright © 2012 The Protein Society.)

The second messenger cyclic di-GMP is a near-ubiquitous signaling molecule that globally alters bacterial cell physiology to promote biofilm formation and community behavior. Much progress was made in recent years towards the identification and characterization of diguanylate cyclases and phosphodiersterases, enzymes involved in the synthesis and degradation of this signaling compound. In contrast, our knowledge of the nature and mechanistic details of c-di-GMP effector proteins lags behind, primarily because effective tools for their specific enrichment and rapid analysis are missing. In this report we demonstrate that a novel tri-functional c-di-GMP-specific Capture Compound (cdG-CC) can be effectively used to identify and validate c-di-GMP binding proteins. The cdG-CC was able to specifically and efficiently pull down bona fide c-di-GMP effector proteins. Furthermore, in combination with mass spectrometry (CCMS), this technology robustly identified a substantial fraction of the known c-di-GMP signaling components directly from cell extracts of different model organisms. Finally, we applied the CCMS technique to profile c-di-GMP binding proteins of Pseudomonas aeruginosa and Salmonella enterica serovar typhimurium. Our studies establish CCMS as a powerful and versatile tool to identify and analyze components of the cellular c-di-GMP pathway in a wide range of different organisms., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mutations in PHF6 are the cause of Börjeson-Forssman-Lehman syndrome (BFLS), an X-linked intellectual disability (XLID) disorder, and both T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). The PHF6 gene encodes a protein with two plant homeodomain (PHD)-like zinc finger domains. As many PHD-like domains function to target chromatin remodelers to post-translationally modified histones, this suggests a role for PHF6 in chromatin regulation. However, PHD domains are usually found in association with a catalytic domain, a feature that is lacking in PHF6. This distinct domain structure and the minimal information on its cellular function prompted us to perform a proteomic screen to identify PHF6 binding partners. We expressed recombinant Flag-tagged PHF6 in HEK 293T cells for coimmunoprecipitation, and analyzed the purified products by mass spectrometry. We identified proteins involved in ribosome biogenesis, RNA splicing, and chromatin regulation, consistent with PHF6 localization to both the nucleoplasm and nucleolus. Notably, PHF6 copurified with multiple constituents of the nucleosome remodeling and deacetylation (NuRD) complex, including CHD4, HDAC1, and RBBP4. We demonstrate that this PHF6-NuRD complex is not present in the nucleolus but is restricted to the nucleoplasm. The association with NuRD represents the first known interaction for PHF6 and implicates it in chromatin regulation.

Since LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications over the past decade, numerous studies have performed relative and/or absolute abundance determinations across large sets of proteins. In this study, we discovered prognostic biomarker candidates from limited breast cancer tissue samples using discovery-through-verification strategy combining iTRAQ method followed by selected reaction monitoring/multiple reaction monitoring analysis (SRM/MRM). We identified and quantified 5122 proteins with high confidence in 18 patient tissue samples (pooled high-risk (n=9) or low-risk (n=9)). A total of 2480 proteins (48.4%) of them were annotated as membrane proteins, 16.1% were plasma membrane and 6.6% were extracellular space proteins by Gene Ontology analysis. Forty-nine proteins with >2-fold differences in two groups were chosen for further analysis and verified in 16 individual tissue samples (high-risk (n=9) or low-risk (n=7)) using SRM/MRM. Twenty-three proteins were differentially expressed among two groups of which MFAP4 and GP2 were further confirmed by Western blotting in 17 tissue samples (high-risk (n=9) or low-risk (n=8)) and Immunohistochemistry (IHC) in 24 tissue samples (high-risk (n=12) or low-risk (n=12)). These results indicate that the combination of iTRAQ and SRM/MRM proteomics will be a powerful tool for identification and verification of candidate protein biomarkers.

It is postulated that insect pheromone-binding proteins (PBPs) are involved in sex pheromone reception, while the general odorant-binding proteins (GOBPs) are involved in reception of the general odorants including plant volatiles. However, this functional specificity is not completely conclusive. In the present study, full-length sequences of two new OBP genes were molecularly identified as OachPBP1 and OachGOBP2 from Orthaga achatina, an important pest of the camphor tree Cinnamomum camphora. Quantification of transcript levels by qRT-PCR showed that the two genes highly expressed in antennae, with OachPBP1 male-biased and OachGOBP2 similar between sexes. These expression patterns are consistent with the generally proposed functions of PBPs and GOBPs. With the recombinant proteins obtained by a bacterial expression system, the binding specificity of these proteins was further investigated and compared using the competitive binding assay. OachPBP1 exhibited high binding affinities with all three putative sex pheromones and 10 pheromone analogs, supporting its role in pheromone reception. On the other hand, in addition to binding with some plant volatiles, OachGOBP2 surprisingly displayed similar or even higher binding affinities with the sex pheromones than OachPBP1. Therefore, we propose that OachGOBP2 might play roles in reception of sex pheromone. Additionally, plant volatiles farnesol and farnesene showed high binding with both OachGOBP2 and OachPBP1, suggesting that these volatile chemicals have regulatory functions in the behavior of O. achatina., (© 2012 Wiley Periodicals, Inc.)

Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)(10). With Gly-Pro-Cys triplets appended to both of its termini, designated CRP(cys), chemical cross-linking using heterobifunctional reagents generates CRP(cys)-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP(cys) antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The D-alanyl lipoteichoic acids (D-alanyl LTAs) present in the cell walls of Gram-positive bacteria play crucial roles in autolysis, cation homeostasis and biofilm formation. The alanylation of LTAs requires the D-alanyl carrier protein DltC to transfer D-Ala onto a membrane-associated LTA. Here, DltC from Staphylococcus epidermidis (SeDltC) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.83 Å and belonged to space group P2, with unit-cell parameters a = 66.26, b = 53.28, c = 88.05 Å, β = 98.22°. The results give a preliminary crystallographic analysis of SeDltC and shed light on the functional role of DltC in the alanylation of LTAs.

Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 Å. The crystal belonged to space group C222(1), with unit-cell parameters of a=69.395, b=199.572, c=131.673 Å, and α=β=γ=90°., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The PEBP (phosphatidylethanolamine-binding protein) family is a large group of proteins whose human member, hPEBP1, has been shown to play multiple functions, influencing intracellular signaling cascades, cell cycle regulation, neurodegenerative processes, and reproduction. It also acts, by an unknown mechanism, as a metastasis suppressor in a number of cancers. A more complete understanding of its biological role is thus necessary. As the yeast Saccharomyces cerevisiae is a powerful and easy to handle model organism, we focused on Tfs1p, the yeast ortholog of hPEBP1. In a previous study based on a two-hybrid approach, we showed that Tfs1p interacts and inhibits Ira2p, a GTPase Activating Protein (GAP) of the small GTPase Ras. To further characterize the molecular functions of Tfs1p, we undertook the identification of protein complexes formed around Tfs1p using a targeted proteomics approach. Complexed proteins were purified by tandem-affinity, cleaved with trypsin, and identified by nanoflow liquid chromatography coupled with tandem mass spectrometry. Overall, 14 new interactors were identified, including several proteins involved in intermediate metabolism. We confirmed by co-immunoprecipitation that Tfs1p interacts with Glo3p, a GAP for Arf GTPases belonging to the Ras superfamily of small GTPases, indicating that Tfs1p may be involved in the regulation of another GAP. We similarly confirmed the binding of Tfs1p with the metabolic enzymes Idp1p and Pro1p. Integration of these results with known functional partners of Tfs1p shows that two subnetworks meet through the Tfs1p node, suggesting that it may act as a bridge between cell signaling and intermediate metabolism in yeast.

Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture.

During heat shock stress, importin β family-mediated nucleocytoplasmic trafficking is downregulated, whereas nuclear import of the molecular chaperone Hsp70s is upregulated. Here, we identify a nuclear import pathway that operates during heat shock stress and is mediated by an evolutionarily conserved protein named "Hikeshi," which does not belong to the importin β family. Hikeshi binds to FG-Nups and translocates through nuclear pores on its own, showing characteristic features of nuclear transport carriers. In reconstituted transport, Hikeshi supports the nuclear import of the ATP form of Hsp70s, but not the ADP form, indicating the importance of the Hsp70 ATPase cycle in the import cycle. In living cells, depletion of Hikeshi inhibits heat shock-induced nuclear import of Hsp70s, reduces cell viability after heat shock stress, and significantly delays the attenuation and reversion of multiple heat shock-induced nuclear phenotypes. Nuclear Hsp70s rescue the effect of Hikeshi depletion at least in part. Thus, Hsp70s counteract heat shock-induced damage by acting inside of the nucleus., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Secretion of the Hedgehog morphogen induces different cell fates over the short and long ranges during developmental patterning. Mature Hedgehog carries hydrophobic palmitic acid and cholesterol modifications essential for its correct spread. The long-range activity of Hedgehog raises questions about how a dually lipidated protein can spread in the hydrophilic environment of the extracellular space. There is compelling experimental evidence in favour of the existence of several different carriers for Hedgehog transportation, via very different routes. This suggests that different accessory proteins and cellular machineries may be involved in the specific release of Hedgehog. I suggest that Hh carriers may work in parallel within a given cell and that developmental context may condition the choice of Hh carrier in secreting cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Fanconi anemia (FA) is a chromosomal instability disorder associated with deficiencies in the Fanconi anemia complementation group (FANC) network. A complex consisting of FANCM-associated histone-fold proteins 1 and 2 (MHF1 and MHF2) has been shown to act cooperatively with FANCM in DNA damage repair in the FA pathway. Here we report the structure of Saccharomyces cerevisiae MHF complex in which MHF1 and MHF2 assume a typical histone fold, and the complex has a heterotetrameric architecture similar to that of the histones (H3-H4)₂ heterotetramer. Loop L2 of MHF1 is probably involved in DNA binding, and loop L3 and helices α2 and α3 of one MHF1 subunit interact with those of the other to form two heterotetramer interfaces. Further genetic data demonstrate that the heterotetramer assembly is essential for the function of the complex in DNA repair. These results provide, to the best of our knowledge, new mechanistic insights into the function of the MHF complex., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ(54) co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH(4)Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Overexpression of recombinant N-acetyl-D-glucosamine 2-epimerase, one of the key enzymes for the synthesis of N-acetylneuraminic acid, in E. coli led to the formation of protein inclusion bodies. In this study we report the recovery of active epimerase from inclusion bodies by direct solubilization with Tris buffer. At pH 7.0, 25% of the inclusion bodies were solubilized with Tris buffer. The specific activity of the solubilized proteins, 2.08±0.02 U/mg, was similar to that of the native protein, 2.13±0.01 U/mg. The result of circular dichroism spectroscopy analysis indicated that the structure of the solubilized epimerase obtained with pH 7.0 Tris buffer was similar to that of the native epimerase purified from the clarified cell lysate. As expected, the extent of deviation in CD spectra increased with buffer pH. The total enzyme activity recovered by solubilization from inclusion bodies, 170.41±10.06 U/l, was more than 2.5 times higher than that from the clarified cell lysate, 67.32±5.53 U/l. The results reported in this study confirm the hypothesis that the aggregation of proteins into inclusion bodies is reversible and suggest that direct solubilization with non-denaturing buffers is a promising approach for the recovery of active proteins from inclusion bodies, especially for aggregation-prone multisubunit proteins., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The intracellular transport of lipids from the sites of their synthesis to their appropriate destination is a critical step for lipid metabolism. One well-defined inter-organelle lipid movement is the transport of ceramide by ceramide transport protein (CERT). Ceramide, a key intermediate for both sphingomyelin and glycosphingolipids, is synthesized at the endoplasmic reticulum and delivered to the Golgi apparatus to be converted to sphingomyelin. CERT delivers ceramide from the ER to the Golgi apparatus in a non-vesicular and ATP-dependent manner. This chapter describes a reconstitution assay system for ceramide transport with semi-intact cells, which is useful for the study of the CERT-mediated inter-organelle transport of ceramide., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cystine-knot miniproteins, also known as knottins, contain a conserved core of three tightly woven disulfide bonds which impart extraordinary thermal and proteolytic stability. Interspersed between their conserved cysteine residues are constrained loops that possess high levels of sequence diversity among knottin family members. Together these attributes make knottins promising molecular scaffolds for protein engineering and translational applications. While naturally occurring knottins have shown potential as both diagnostic agents and therapeutics, protein engineering is playing an important and increasing role in creating designer molecules that bind to a myriad of biomedical targets. Toward this goal, rational and combinatorial approaches have been used to engineer knottins with novel molecular recognition properties. Here, methods are described for creating and screening knottin libraries using yeast surface display and fluorescence-activated cell sorting. Protocols are also provided for producing knottins by synthetic and recombinant methods, and for measuring the binding affinity of knottins to target proteins expressed on the cell surface., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Carrier proteins (CPs) are integral components of fatty acid synthases, polyketide synthases, and nonribosomal peptide synthetases and play critical roles in the biosynthesis of fatty acids, polyketides, and nonribosomal peptides. An emerging role CPs play in natural product biosynthesis involves tailoring enzymes that act on CP-tethered substrates. These enzymes provide a new opportunity to engineer natural product diversity by exploiting CPs to increase substrate promiscuity for the tailoring steps. This chapter describes protocols for in vitro biochemical characterization of SgcC3 and SgcC that catalyze chlorination and hydroxylation of SgcC2-tethered (S)-β-tyrosine and analogues in the biosynthesis of the enediyne chromophore of the chromoprotein C-1027. These protocols are applicable to mechanistic characterization and engineered exploitation of other tailoring enzymes that act on CP-tethered substrates in natural product biosynthesis and structural diversification. The ultimate goal is to use the in vitro findings to guide in vivo engineering of designer natural products., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors from plants confer resistance to fungal pathogens and many other pathogenic organisms. Their low expression makes it challenging to purify these receptors from plants in sufficient quantities to be able to identify interacting proteins by mass spectrometry. Here we describe a protocol to affinity-purify recombinant NB-LRR immune receptors, fused to the streptavidin-binding peptide tag.

Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity.

Intrinsically disordered proteins (IDPs) differ from ordered proteins at several levels: structural, functional, and conformational. Amino acid biases also drive atypical responses of IDPs to changes in their environment. Among several specific features, the conformational behavior of IDPs is characterized by the low cooperativity (or the complete lack thereof) of the denaturant-induced unfolding. In fact, the denaturant-induced unfolding of native molten globules can be described by shallow sigmoidal curves, whereas urea- or guanidinium hydrochloride-induced unfolding of native pre-molten globules or native coils is a noncooperative process and typically is seen as monotonous feature-less changes in the studied parameters. This chapter describes some of the most characteristic features of the IDP conformational behavior.

We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap(4)A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap(4)A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap(4)A-binding partners, and we have expectations that others can be found with further technical improvements in our methods., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or expensive antibody preparations.

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

Group A streptococcus (GAS) harbours several virulence factors, including M protein (coded by the emm gene) and superantigens (SAgs). SAgs are extracellular toxins that directly activate the immune system by cross-binding to the HLA class II molecule and T cell receptor (TCR), thereby causing activation of up to 30% of the T cells and subsequent massive secretion of cytokines. Forty-eight GAS strains isolated from patients at Norwegian hospitals between 1988 and 2004 were included in this study. Of these, 24 were invasive streptococcal toxic shock syndrome (STSS) or necrotizing fasciitis (NF) isolates and 24 were non-invasive pharyngitis isolates, matched for having the same T-type and year of isolation as the invasive isolates. The isolates were characterized by emm sequence typing, multilocus sequence typing (MLST) and SAg gene profiles. A correlation between T-type, emm type, sequence type and SAg gene profile was revealed. No difference between invasive and non-invasive isolates regarding serotype or genotype was demonstrated. Selected invasive and non-invasive isolates with identical SAg gene profiles were analysed for SAg activity in bacterial growth culture media with and without human cell culture media added. A human T cell proliferation assay was used as measurement for SAg activity and simultaneously we also measured the cytokine content in normal human peripheral blood leucocyte cell culture media. The results revealed that invasive and non-invasive isolates did not differ significantly in SAg activity as it is present in semipurified bacterial culture medium., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

Mechanically recovered meat is cheaper than raw meat and thus has been incorporated into many meat-derived products. EU regulations exclude mechanically recovered meat from the definition of meat; as a consequence analytical procedures are needed to differentiate it from hand-deboned meat. The present pilot study has utilized a proteomic approach to find potential markers for the detection of chicken mechanically recovered meat. Intact proteins were extracted from raw meat and then analyzed with OFF-GEL electrophoresis followed by SDS-PAGE and identification of potential markers by nano-LC-MS/MS. It was shown that it is possible to extract, separate and identify key proteins from processed meat material. Potential chicken mechanically recovered meat markers--hemoglobin subunits and those similar to myosin-binding protein C were also identified., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cell metabolic processes are constantly producing reactive oxygen species (ROS), which have deleterious effects by triggering, for example, DNA damage. Numerous enzymes such as catalase, and small compounds such as vitamin C, provide protection against ROS. The TLDc domain of the human oxidation resistance protein has been shown to be able to protect DNA from oxidative stress; however, its mechanism of action is still not understood and no structural information is available on this domain. Structural information on the TLDc domain may therefore help in understanding exactly how it works. Here, the purification, crystallization and preliminary crystallographic studies of the TLDc domain from zebrafish are reported. Crystals belonging to the orthorhombic space group P2(1)2(1)2 were obtained and diffracted to 0.97 Å resolution. Selenomethionine-substituted protein could also be crystallized; these crystals diffracted to 1.1 Å resolution and the structure could be solved by SAD/MAD methods., (© 2011 International Union of Crystallography. All rights reserved.)

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN) , HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair., (© 2011 The Authors Journal compilation © 2011 FEBS.)

A specific fatty acid binding protein was isolated from Giardia lamblia, using an affinity column with butyric acid acting as a ligand in place of stearic acid. This method has proved to be more efficient than the one previously described using stearic acid as ligand. The purified fraction showed 8 electrophoretic bands of proteins, with molecular weights ranging between 8 and 80 kDa. This pattern is a consequence of the aggregation of a protein with a molecular weight of 8,215 Da, corresponding to the lower molecular weight band, the only one capable of binding to fatty acids. The labeled oleic acid bound to these purified proteins was replaced by a 100-fold greater concentration of taurocholate, glycocholate, deoxycholate, palmitic acid, and arachidonic acid, having a greater displacement of the bile salts than the free fatty acids.

Chromium was proposed to be an essential element over 50 y ago and was shown to have therapeutic potential in treating the symptoms of type 2 diabetes; however, its mechanism of action at a molecular level is unknown. One chromium-binding biomolecule, low-molecular weight chromium-binding substance (LMWCr or chromodulin), has been found to be biologically active in in vitro assays and proposed as a potential candidate for the in vivo biologically active form of chromium. Characterization of the organic component of LMWCr has proven difficult. Treating bovine LMWCr with trifluoroacetic acid followed by purification on a graphite powder micro-column generates a heptapeptide fragment of LMWCr. The peptide sequence of the fragment was analyzed by MS and tandem MS (MS/MS and MS/MS/MS) using collision-induced dissociation and post-source decay. Two candidate sequences, pEEEEGDD and pEEEGEDD (where pE is pyroglutamate), were identified from the MS/MS experiments; additional tandem MS suggests the sequence is pEEEEGDD. The N-terminal glutamate residues explain the inability to sequence LMWCr by the Edman method. Langmuir isotherms and Hill plots were used to analyze the binding constants of chromic ions to synthetic peptides similar in composition to apoLMWCr. The sequence pEEEEGDD was found to bind 4 chromic ions per peptide with nearly identical cooperativity and binding constants to those of apoLMWCr. This work should lead to further studies elucidating or eliminating a potential role for LMWCr in treating the symptoms of type 2 diabetes and other conditions resulting from improper carbohydrate and lipid metabolism.

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1) . Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein's biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1) , showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of α-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian α-amylase activity in vitro., (Copyright © Physiologia Plantarum 2011.)

We recently reported a one-step affinity purification method using a silica-binding protein, designated Si-tag, as a fusion partner and silica particles as the specific adsorbents (Ikeda et al., Protein Expr. Purif. 71 [2010] 91-95) [13]. In this study, we demonstrate that the Si-tag also binds to the silica surface even under denaturing conditions, thereby facilitating affinity purification of recombinant proteins from inclusion bodies. A fusion protein of the Si-tag and a biotin acceptor peptide (AviTag), which was expressed as inclusion bodies in Escherichia coli, was used as a model protein. To simplify our purification method, we disrupted recombinant E. coli cells by sonication in the presence of 8M urea with concomitant solubilization of the inclusion bodies. The fusion protein was recovered with a purity of 90 ± 3% and yield of 92 ± 6% from the cleared cell lysate. We also discuss the binding mechanism of the Si-tag to a silica surface in the presence of high concentrations of denaturant. We propose that the intrinsic disorder of the polycationic Si-tag polypeptide plays an important role in its binding to the silica surface under denaturing conditions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Previously we have shown that extracts from Xenopus egg jelly (egg water) increase the passage of sperm through a porous membrane in a dose-dependent manner. Although this assay has shown that sperm accumulation occurs only in the presence of an egg water gradient, it has not revealed the dynamic features of how Xenopus sperm swim in such gradients. Here, we use video microscopic observations to trace sperm trajectories in a Zigmond chamber. Our results show that Xenopus sperm swim in linear and gently curving paths and only infrequently perform turns. In the presence of an egg water gradient, however, the percent of sperm swimming up the gradient axis and the net distance traveled by each sperm along this axis was increased significantly. There was no change in curvilinear velocity. Rather, the orientation of sperm travel was shifted to more closely match that of the gradient axis. In addition, using a porous filter assay, we demonstrate that the egg water protein allurin, in both purified and recombinant forms, stimulates directed motility of sperm. Finally, we use Oregon Green 488-conjugated allurin to show that this protein binds primarily to the sperm midpiece; binding of allurin to the entire head was observed in a minor subpopulation of sperm. Dose dependence of allurin binding occurred over the 0-1 µg/ml range and correlated well with previously published dose-dependent sperm attraction data. Binding was rapid with a half-time of about 10 sec. These data suggest that egg water proteins bind to sperm and modify sperm-orienting behavior., (Copyright © 2011 Wiley-Liss, Inc.)

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.