Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins.

Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr(4+), afforded better immobilization of the peptides and proteins and efficient capture of their targets.

In vivo phosphorylation of a peptide tag for protein purification.

OBJECTIVES: To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag. RESULTS: The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III). CONCLUSION: The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

Design and optimization of a phosphopeptide anchor for specific immobilization of a capture protein on zirconium phosphonate modified supports.

The attachment of affinity proteins onto zirconium phosphonate coated glass slides was investigated by fusing a short phosphorylated peptide sequence at one extremity to enable selective bonding to the active surface via the formation of zirconium phosphate coordinate covalent bonds. In a model study, the binding of short peptides containing zero to four phosphorylated serine units and a biotin end-group was assessed by surface plasmon resonance-enhanced ellipsometry (SPREE) as well as in a microarray format using fluorescence detection of AlexaFluor 647-labeled streptavidin. Significant binding to the zirconated surface was only observed in the case of the phosphopeptides, with the best performance, as judged by streptavidin capture, observed for peptides with three or four phosphorylation sites and when spotted at pH 3. When fusing similar phosphopeptide tags to the affinity protein, the presence of four phosphate groups in the tag allows efficient immobilization of the proteins and efficient capture of their target.

New Phosphorylation Sites of Rad51 by c-Met Modulates Presynaptic Filament Stability.

Genomic instability through deregulation of DNA repair pathways can initiate cancer and subsequently result in resistance to chemo and radiotherapy. Understanding these biological mechanisms is therefore essential to overcome cancer. RAD51 is the central protein of the Homologous Recombination (HR) DNA repair pathway, which leads to faithful DNA repair of DSBs. The recombinase activity of RAD51 requires nucleofilament formation and is regulated by post-translational modifications such as phosphorylation. In the last decade, studies have suggested the existence of a relationship between receptor tyrosine kinases (RTK) and Homologous Recombination DNA repair. Among these RTK the c-MET receptor is often overexpressed or constitutively activated in many cancer types and its inhibition induces the decrease of HR. In this study, we show for the first time that c-MET is able to phosphorylate the RAD51 protein. We demonstrate in vitro that c-MET phosphorylates four tyrosine residues localized mainly in the subunit-subunit interface of RAD51. Whereas these post-translational modifications do not affect the presynaptic filament formation, they strengthen its stability against the inhibitor effect of the BRC peptide obtained from BRCA2. Taken together, these results confirm the role of these modifications in the regulation of the BRCA2-RAD51 interaction and underline the importance of c-MET in DNA damage response.

Site-Specific Conjugation for Fully Controlled Glycoconjugate Vaccine Preparation.

Glycoconjugate vaccines are formed by covalently link a carbohydrate antigen to a carrier protein whose role is to achieve a long lasting immune response directed against the carbohydrate antigen. The nature of the sugar antigen, its length, its ratio per carrier protein and the conjugation chemistry impact on both structure and the immune response of a glycoconjugate vaccine. In addition it has long been assumed that the sites at which the carbohydrate antigen is attached can also have an impact. These important issue can now be addressed owing to the development of novel chemoselective ligation reactions as well as techniques such as site-selective mutagenesis, glycoengineering, or extension of the genetic code. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. The preparation and characterization of homogeneous bivalent pneumococcal vaccines is reported. A synthetic tetrasaccharide representative of the serotype 14 capsular polysaccharide of Streptococcus pneumoniae has been linked using the thiol/maleimide coupling chemistry to four different Pneumococcal surface adhesin A (PsaA) mutants, each harboring a single cysteine mutation at a defined position. Humoral response of these 1 to 1 carbohydrate antigen/PsaA conjugates have been assessed in mice. Our results showed that the carbohydrate antigen-PsaA connectivity impacts the anti-carrier response and raise questions about the design of glycoconjugate vaccine whereby the protein plays the dual role of immunogen and carrier.

Bivalent Fv antibody fragments obtained by substituting the constant domains of a fab fragment with heterotetrameric molybdopterin synthase.

The antibody Fv fragment is the smallest functional unit of an antibody but for practical use, the VH/VL interface requires stabilization, which is usually accomplished by a peptide linker that joins the two variable domains to form a single chain Fv fragment (scFv). An alternative format to scFv is proposed that (i) allows stabilization of the Fv fragment, and (ii) restores the bivalency of the antibody as a pseudo-F(ab')2 format. This new antibody fragment was constructed by replacing the CHI and CL domains of the Fab fragment with heterotetrameric molybdopterin synthase (MPTS). We found that this format, named MoaFv, improved significantly the cytoplasmic expression of the Fv as a soluble protein in BL21 or Origami Escherichia coli strains. This MoaFv format is expressed as a homogeneous heterotetrameric protein with a Mr value of 110 kDa containing two functional binding sites as revealed by active site titration. In its native condition at 37 degrees C or in the presence of urea, this format was nearly as stable as the corresponding scFv, indicating that non-covalent interactions between the MPTS subunits can replace the covalent peptide linker in scFv. Finally, this MoaFv construct could be a useful format when bivalency is desirable to improve the functional avidity.

Molecular epidemiology of airway colonisation by Aspergillus fumigatus in cystic fibrosis patients.

A total of 109 sequential and multiple Aspergillus fumigatus isolates corresponding to 41 samples from seven cystic fibrosis (CF) patients was typed by random amplification of polymorphic DNA (RAPD) with the primer NS3 from the fungal ribosomal gene 18S subunit, and by sequence-specific DNA primer (SSDP) analysis. RAPD typing of the isolates revealed 10 different genotypes, whereas nine genotypes were identified by SSDP. Combination of the two typing methods permitted the differentiation of 25 overall genotypes. The colonisation typing patterns differed greatly between patients colonised for <1 year by A. fumigatus and long-term colonised patients. Two of three recently colonised patients presented a large number of types even in the same sample, unlike the chronically colonised patients, who harboured a limited number of genotypes. In the latter, the occurrence of a dominant genotype, usually the overall genotype 2, tended to reflect to the duration of colonisation. Moreover, anti-catalase antibodies to A. fumigatus appeared in most cases to be in response to genotype 2. These findings suggest that some strains of A. fumigatus may be selected during prolonged colonisation of the airways in CF patients.

In-vitro resistance to azoles associated with mitochondrial DNA deficiency in Candida glabrata.

A commercially available disk diffusion procedure was used in a large-scale study to evaluate the susceptibility of a wide range of Candida isolates to polyenes and azoles. With almost all isolates of C. glabrata resistant colonies were present within the inhibition zones for the azole compounds fluconazole, ketoconazole and miconazole, and less frequently for isoconazole, econazole and clotrimazole. Ten randomly selected isolates were cloned by limiting dilution and the susceptibility of the resulting strains to polyenes and azoles was determined. All strains presented a similar susceptibility pattern with sensitivity to polyenes and the presence of resistant colonies for all azole compounds except tioconazole. For each strain and each antifungal agent, one of these resistant colonies was subcultured and studied for antifungal susceptibility. All these colonies showed similar properties regardless of which antifungal agent allowed their selection, with increased sensitivity to polyenes and cross-resistance to the azole compounds except tioconazole. Similar results were obtained on Shadomy's modified medium and on synthetic medium. Likewise, determination of MICs by the Etest method confirmed the resistance to fluconazole. Comparative growth studies revealed a respiratory deficiency in the mutants caused by mitochondrial DNA (mtDNA) deletions. In addition, 'petite' mutants were obtained from a wild-type strain by exposure to ethidium bromide, and these respiratory mutants were shown to be resistant to azoles. These results demonstrate the relationship between mtDNA deficiency and resistance to azoles, and provide an interesting model to study the mechanisms of action of these antifungal agents.

Typing of Scedosporium apiospermum by multilocus enzyme electrophoresis and random amplification of polymorphic DNA.

The genetic diversity among epidemiologically unrelated strains of the human pathogenic fungus Scedosporium apiospermum or its teleomorph, Pseudallescheria boydii, from different areas in Europe, was investigated by multilocus enzyme electrophoresis (MLEE) and random amplification of polymorphic DNA (RAPD). Fourteen enzyme activities were analysed by starch gel electrophoresis, corresponding to 27 polymorphic loci and 43 iso-enzymes. Among the enzymes studied, propionate esterase, carboxyl esterase, superoxide dismutase, carbonate dehydratase and malate dehydrogenase were the most polymorphic, allowing the classification of the strains into 6-11 groups each. Combination of the data obtained for the different enzyme activities studied allowed differentiation of the strains. Similarly, a high polymorphism was also revealed by each of the 20 RAPD primers tested, but no single primer was able to differentiate all the strains. The most efficient primers were GC70, UBC-701 and UBC-703, which revealed 17 distinct genotypes each, and combination of the results obtained with this three-primer set allowed complete discrimination of the strains. The dendrograms obtained from MLEE or RAPD by the unweighted pair-group method using arithmetic average cluster analysis did not reveal any clustering according to the geographic origin of the strains or their pathogenicity.

In-vivo selection of an azole-resistant petite mutant of Candida glabrata.

Two isolates of Candida glabrata from the same stool sample from a bone marrow transplant recipient treated with fluconazole, and designated 1084-L for large colonies on yeast extract-peptone-dextrose-agar and 1084-S for small colonies, were analysed. In-vitro susceptibility tests with a commercially available disk diffusion procedure showed that isolate 1084-L had a susceptibility pattern typical of wild-type strains of C. glabrata with sensitivity to polyenes and the presence of resistant colonies randomly distributed within the inhibition zones for all azole compounds except tioconazole. In contrast, isolate 1084-S, which was found by pulsed-field gel electrophoresis and random amplification of polymorphic DNA to be genetically closely related to isolate 1084-L, exhibited cross-resistance to the azole compounds except tioconazole. Determination of MICs by the E-test method confirmed these results, showing that isolate 1084-S had greater sensitivity to amphotericin B and complete resistance to ketoconazole and fluconazole. Growth on agar plates containing glucose or glycerol as the sole carbon source suggested that the resistant isolate had a respiratory deficiency, which was further demonstrated by flow cytometric analysis of the fluorescence of rhodamine 123-stained blastoconidia. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) established the mitochondrial origin of the respiratory deficiency. However, PCR amplification of the mtDNA with primers ML1 and ML6, as well as transmission electron microscopy, suggested a partial deletion of the mtDNA analogous to that described for rho- petite mutants of Saccharomyces cerevisiae. Together, these results provided evidence that the selection of azole-resistant petite mutants of C. glabrata may occur in vivo after fluconazole administration, which might explain, therefore, clinical failure of antifungal therapy.

Evolution of interleukin-15 for higher E. coli expression and solubility.

Directed evolution was used to generate IL-15 mutants with increased solubility and cytoplasmic over-expression in Escherichia coli. A protein solubility selection method was used in which the IL-15 gene was expressed as an N-terminal fusion to chloramphenicol acetyltransferase (CAT) as reporter protein. Clones that grew in the presence of high concentrations of chloramphenicol were then screened by ELISA to assay the binding activity of the IL-15-CAT fusion to the IL-15Rα Sushi domain. Two variants of IL-15, M38 and M253, containing five mutations and one mutation respectively, were selected with a dramatic improvement in solubility; the soluble concentration in cell culture was 12- to 18-fold higher, respectively, than for WT IL-15. Characterization of their binding to IL-15Rα and their ability to stimulate the T-cell growth response showed that M38 binds as strongly as native IL-15 to IL-15Rα and acts as an effective agonist of IL-15.

Genotyping study of Scedosporium apiospermum isolates from patients with cystic fibrosis.

Usually a saprophyte, Scedosporium apiospermum often colonizes the respiratory tracts of patients with cystic fibrosis (CF). In order to improve our understanding of the molecular epidemiology of the airway colonization, 129 sequential and multiple isolates collected from January 1998 to March 1999 from nine CF patients monitored in three hospitals in France were typed by random amplification of polymorphic DNA with primers GC70, UBC-701, and UBC-703. Among these primers, UBC-703 was the most discriminating, allowing the differentiation of 14 genotypes. Combining the results obtained with this three-primer set resulted in the differentiation of 16 genotypes. No common genotype was found among the different patients, and no clustering according to geographic origin of the isolates was seen. In addition, five of the patients were colonized by a single genotype. The others usually exhibited a predominant genotype accompanied by one or two others, which were found occasionally and were genetically close to the predominant genotype. Thus, our study demonstrates the persistence of the fungus despite antifungal treatments and therefore reinforces the need for the development of new antifungals that are more efficient against this species.