Artichoke and Cynar liqueur: Two (not quite) entangled proteomes

Combinatorial peptide ligand libraries (CPLLs) have been adopted to investigate the proteome of artichoke extracts, of a home-made alcoholic infusion and of the Italian Cynar liqueur. The aim of study was not only to perform the deepest investigation so far of the artichoke proteome but also to assess the genuineness of the commercial aperitif via a three-pronged attack. First, different extraction techniques have been used for the characterization of the artichoke's proteome, secondly a home-made infusion has been analyzed and finally the proteome of the commercial drink was checked. The artichoke proteome has been evaluated via prior capture with CPLLs at four different pH (2.2, 4.0, 7.2 and 9.3) values. Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 876 unique gene products in the artichoke extracts, 18 in the home-made infusion and no proteins at all in the Italian Cynar liqueur, casting severe doubts on the procedure stated by the manufacturer (that should be made by an infusion of artichoke leaves plus thirteen different herbs). This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products.     

The sequence of a porcine cDNA encoding α-lactalbumin

A cDNA clone encoding porcine α-lactalbumin (αLA) was isolated and sequenced. The longest clone was 688 nucleotides (nt) long and encoded a preprotein of 141 amino acids (aa) including a leader peptide of 19 aa. The porcine cDNA exhibited a nt similarity of between 72.2%–83.5% to other αLA cDNAs and an aa similarity of between 50.8%–85.2% with other αLA aa sequences. The derived aa sequence varied at three positions from a previously reported sequence for porcine αLA obtained by direct aa sequencing.     

Peptaibiomics: an advanced, rapid and selective analysis of peptaibiotics/peptaibols by SPE/LC-ES-MS

Summary. “Proteomics” and “peptidomics” are used as technical terms to define the analysis and study of all proteins and peptides expressed in an organism or tissue. In analogy we propose the name peptaibiomics for the analysis of a group of fungal peptide antibiotics (peptaibiotics) containing the characteristic amino acid Aib (α-aminoisobutyric acid). In analogy to the peptidome the complete expression of peptaibiotics by fungal multienzyme complexes should be named the peptaibiome. Peptaibiotics are defined as peptides containing Aib and exerting a variety of bioactivities. They comprise the sub-groups of N-acetylated peptaibols, characterized also by a C-terminal amide-linked 2-amino alcohol, and lipopeptaibols having in place of an acetyl group a lipophilic fatty acid acyl group. Furthermore, lipoaminopeptides are also known with long-chain fatty acid on the N-termini, a lipoamino acid in position three and a strongly basic secondary or tertiary amine form a subgroup of mixed forms which could not be integrated in one of these three previously mentioned groups. Here we present a specific and rapid screening method on the peptaibiome applicable directly onto filamentous fungi cultured in a single Petri dish. The method comprises solid-phase extraction (SPE) of peptaibiotics followed by on-line reversed-phase HPLC coupled to an ion trap electrospray tandem mass spectrometer (ES-MS). The presence of these peptides is indicated by characteristic mass differences of Δm = 85.1 Da representing Aib-residues which can be observed in the b-series of acylium fragment ions resulting from ES-MS. Partial sequences can be deduced from the data and compared with structures compiled in electronic peptaibol data bases. The judgement is possible whether or not structures are novel, already known or related to known structures. Suitability of the method is demonstrated with the analysis of strains of Trichoderma and its teleomorph Hypocrea. New sequences of peptaibiotics are presented and those being related to established 10- to 18-residue peptaibols trichovirin, trichogin and trichotoxin, which have been described in the literature.     

A new generation of protein display scaffolds for molecular recognition

Engineered antibodies and their fragments are invaluable tools for a vast range of biotechnological and pharmaceutical applications. However, they are facing increasing competition from a new generation of protein display scaffolds, specifically selected for binding virtually any target. Some of them have already entered clinical trials. Most of these nonimmunoglobulin proteins are involved in natural binding events and have amazingly diverse origins, frameworks, and functions, including even intrinsic enzyme activity. In many respects, they are superior over antibody-derived affinity molecules and offer an ever-extending arsenal of tools for, e.g., affinity purification, protein microarray technology, bioimaging, enzyme inhibition, and potential drug delivery. As excellent supporting frameworks for the presentation of polypeptide libraries, they can be subjected to powerful in vitro or in vivo selection and evolution strategies, enabling the isolation of high-affinity binding reagents. This article reviews the generation of these novel binding reagents, describing validated and advanced alternative scaffolds as well as the most recent nonimmunoglobulin libraries. Characteristics of these protein scaffolds in terms of structural stability, tolerance to multiple substitutions, ease of expression, and subsequent applications as specific targeting molecules are discussed. Furthermore, this review shows the close linkage between these novel protein tools and the constantly developing display, selection, and evolution strategies using phage display, ribosome display, mRNA display, cell surface display, or IVC (in vitro compartmentalization). Here, we predict the important role of these novel binding reagents as a toolkit for biotechnological and biomedical applications.     

Developing bifunctional β‐lactamase molecules with built‐in target‐recognizing module for prodrug therapy: identification of Enterobacter Cloacae P99 cephalosporinase loops suitable for randomization and phage‐display selection

This study was focused on developing catalytically active β‐lactamase enzyme molecules that have target‐recognizing sites built within their scaffold. Using phage‐display approach, nine libraries were constructed by inserting the randomized linear or cysteine‐constrained heptapeptides in the five different loops on the outer surface of P99 β‐lactamase molecule. The pIII signal peptide of Sec‐pathway was employed for a periplasmic translocation of the β‐lactamase fusion protein, which we found more efficient than the DsbA signal peptide of SRP‐pathway. The randomized heptapeptide loops replaced native amino acids between positions ³⁴Y‐³⁷K, ²³⁸M‐²⁴⁶A, ²⁷⁵N‐²⁸⁰A, ³⁰⁵A‐³¹¹S, or ³²⁹I‐³³⁴I of the P99 β‐lactamase molecules for generating the loop‐1 to ‐5 libraries, respectively. The diversity of each loop library was judged by counting the primary and β‐lactamase‐active clones. The linear peptide inserts in the loop‐2 library showed the maximum number of the β‐lactamase‐active clones, followed by the loop‐5, loop‐3, and loop‐4. The insertion of the cysteine‐constrained loops exhibited a dramatic loss of the enzyme‐active β‐lactamase clones. The complexity of the loop‐2 linear library, as determined by the frequency and diversity of amino acid distributions in the randomized region, appears consistent with the standards of other types of phage display library systems. The selection of the loop‐2 linear library on streptavidin protein as a test target identified several β‐lactamase clones that specifically bound to streptavidin. In conclusion, this study identified the suitability of the loop‐2 of P99 β‐lactamase for constructing a phage‐display library of the β‐lactamase enzyme‐active molecules that can be selected against a target. This is an enabling step in our long‐term goal of developing bifunctional β‐lactamase molecules against cancer‐specific targets for enzyme prodrug therapy of cancer. Copyright © 2009 John Wiley & Sons, Ltd.     

Metagenomic gene discovery: How far have we moved into novel sequence space?

Metagenomics emerged in the late 1990s as a tool for accessing and studying the collective microbial genetic material in the environment. The advent of the technology generated great excitement, as it has provided new opportunities and technologies for studying the wealth of microbial genetic diversity in the environment. Metagenomics has been widely predicted to access new dimensions of protein sequence space. A decade on, we review how far we have actually moved into new sequence space (and other aspects of protein space) using metagenomic tools. While several novel enzyme activities and protein structures have been identified through metagenomic strategies, the greatest advancement has been made in the isolation of novel protein sequences, some of which have no close relatives, form deeply branched lineages and even represent novel families. This is particularly true for glycosyl hydrolases and lipase/esterases, despite the fact that these activities are frequently screened for in metagenomic studies. However, there is much room for improvement in the methods employed and they will need to be addressed so that access to novel biocatalytic activities can be widened.     

PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries

ABSTRACT. Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray–Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.     

Inhibition of Aβ42 aggregation using peptides selected from combinatorial libraries

Increasing evidence suggests that the aggregation of the small peptide Aβ42 plays an important role in the development of Alzheimer's disease. Inhibiting the initial aggregation of Aβ42 may be an effective treatment for preventing, or slowing, the onset of the disease. Using an in vivo screen based on the enzyme EGFP, we have searched through two combinatorially diverse peptide libraries to identify peptides capable of inhibiting Aβ42 aggregation. From this initial screen, three candidate peptides were selected and characterized. ThT studies indicated that the selected peptides were capable of inhibiting amyloid aggregation. Additional ThT studies showed that one of the selected peptides was capable of disaggregating preformed Aβ42 fibers. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Phylogenetic characterization of the heterotrophic bacterial communities inhabiting a marine recirculating aquaculture system

Aims: The aim of the present work was to characterize the heterotrophic bacterial community of a marine recirculating aquaculture system (RAS). Methods and Results: An experimental RAS was sampled for the rearing water (RW) and inside the biofilter. Samples were analysed for bacterial abundances, community structure and composition by using a combination of culture‐dependent and ‐independent techniques. The most represented species detected among biofilter clones was Pseudomonas stutzeri, while Ruegeria spp. and Roseobacter spp. were more abundant among isolates. In comparison, the genera Roseobacter and Ruegeria were well represented in both the biofilter and the RW samples. A variety of possible bacterial pathogens (e.g. Vibrio spp., Erwinia spp. and Coxiella spp.) were also identified in this study. Conclusions: Results revealed that the bacterial community in the RW was quite different to that associated with the biofilter. Moreover, data obtained suggest that the whole bacterial community can be involved in maintaining an effective and a stable rearing environment (shelter effect). Significance and Impact of the Study: Improving the reliability and the sustainability of RAS depends on the correct management of the bacterial populations inside it. This study furnishes more accurate information on the bacterial populations and better clarifies the existing relationships between the bacterial flora in the RW and that associated with the biofilter.