Mariela M. Marani

Screening of One-Bead-One-Peptide Combinatorial Library Using Red Fluorescent Dyes. Presence of Positive and False Positive Beads

To screen one-bead-one-compound (OBOC) combinatorial libraries, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with this molecule are then identified and isolated for compound structure determination. Here we describe an OBOC peptide library screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using the COPAS BIO-BEAD flow sorting equipment to separate fluorescent from nonfluorescent beads. The red dyes used were ATTO 590 and Texas Red. After incubating the library with the SA-red fluorescent dye conjugate, we isolated positive beads caused by peptide-SA interaction and false positive beads produced by peptide fluorescent dye interaction. These false positives were a drawback when sorting beads by COPAS. However,an in depth analysis of both kinds of beads allowed the differentiation of positives from false positives. The false positive beads showed bright homogeneous fluorescence, while positive beads had a heterogeneous fluorescence, exhibiting a characteristic halo appearance, with fluorescence intensity greatest at the bead surface and lowest in the core. The difference was more evident when using Texas Red instead of ATTO 590. Thus, positive beads could be manually separated from false positive ones. The beads were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Most of the sequences obtained from positive beads had the His-Pro-Gln motif. Peptides from false positive beads were rich in Leu/Ileu, His, Phe, and Tyr.

Characterization and Biological Activities of Ocellatin Peptides from the Skin Secretion of the Frog Leptodactylus pustulatus

Eight new peptides were isolated from the skin secretion of the frog Leptodactylus pustulatus and their amino acid sequences determined by de novo sequencing and by cDNA cloning. Structural similarities between them and other antimicrobial peptides from the skin secretion of Leptodactylus genus frogs were found. Ocellatins-PT1 to -PT5 (25 amino acid residues) are amidated at the C-terminus, while ocellatins-PT6 to -PT8 (32 amino acid residues) have free carboxylates. Antimicrobial activity, hemolytic tests, and cytotoxicity against a murine fibroblast cell line were investigated. All peptides, except for ocellatin-PT2, have antimicrobial activity against at least one Gram-negative strain. Ocellatin-PT8 inhibited the growth of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella choleraesuis strains with MICs in the 60-240 μM range. No significant effect was observed in human erythrocytes and in a murine fibroblast cell line after exposure to the peptides at MICs. A comparison between sequences obtained by both direct HPLC-MS de novo sequencing and cDNA cloning demonstrates the secretion of mature peptides derived from a pre-pro-peptide structure.

Affinity chromatography based on a combinatorial strategy for rerythropoietin purification.

Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively.

Sample preparation for sequencing hits from one-bead-one-peptide combinatorial libraries by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Optimization of bead analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after the screening of one-bead-one-peptide combinatorial libraries was achieved, involving the fine-tuning of the whole process. Guanidine was replaced by acetonitrile (MeCN)/acetic acid (AcOH)/water (H(2)O), improving matrix crystallization. Peptide-bead cleavage with NH(4)OH was cheaper and safer than, yet as efficient as, NH(3)/tetrahydrofuran (THF). Peptide elution in microtubes instead of placing the beads in the sample plate yielded more sample aliquots. Successive dry layers deposit sample preparation was better than the dried droplet method. Among the matrices analyzed, alpha-cyano-4-hydroxycinnamic acid resulted in the best peptide ion yield. Cluster formation was minimized by the addition of additives to the matrix.

Peptide Affinity Chromatography Based on Combinatorial Strategies for Protein Purification

We describe a method to develop affinity chromatography matrices with short peptide ligands for protein purification. The method entitles the following: (a) synthesis of a combinatorial library on the hydromethylbenzoyl (HMBA)-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry, (b) library screening with the protein of interest labeled with a fluorescent dye or biotin, (c) identification of peptides contained on positive beads by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), (d) solid-phase peptide ligand synthesis and immobilization in chromatographic supports, and (e) evaluation of protein adsorption on peptide affinity matrices from the equilibrium isotherms and breakthrough curves.

Layer-by-layer films containing peptides of the Cry1Ab16 toxin from Bacillus thuringiensis for potential biotechnological applications

Cry1Ab16 is a toxin of crystalline insecticidal proteins that has been widely used in genetically modified organisms (GMOs) to gain resistance to pests. For the first time, in this study, peptides derived from the immunogenic Cry1Ab16 toxin (from Bacillus thuringiensis) were immobilized as layer-by-layer (LbL) films. Given the concern about food and environmental safety, a peptide with immunogenic potential, PcL342-354C, was selected for characterization of the electrochemical, optical, and morphological properties. The results obtained by cyclic voltammetry (CV) showed that the peptide have an irreversible oxidation process in electrolyte of 0.1 mol · L(-1) potassium phosphate buffer (PBS) at pH7.2. It was also observed that the electrochemical response of the peptide is governed mainly by charge transfer. In an attempt to maximize the electrochemical signal of peptide, it was intercalated with natural (agar, alginate and chitosan) or synthetic polymers (polyethylenimine (PEI) and poly(sodium 4-styrenesulfonate (PSS)). The presence of synthetic polymers on the film increased the electrochemical signal of PcL342-354C up to 100 times. Images by Atomic Force Microscopy (AFM) showed that the immobilized PcL342-354C formed self-assembled nanofibers with diameters ranging from 100 to 200 nm on the polymeric film. By UV-Visible spectroscopy (UV-Vis) it was observed that the ITO/PEI/PSS/PcL342-354C film grows linearly up to the fifth layer, thereafter tending to saturation. X-ray diffraction confirmed the presence on the films of crystalline ITO and amorphous polypeptide phases. In general, the ITO/PEI/PSS/PcL342-354C film characterization proved that this system is an excellent candidate for applications in electrochemical sensors and other biotechnological applications for GMOs and environmental indicators.

In silico peptide prediction for antibody generation to recognize 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in genetically modified organisms

For the prospective immunorecognition of 5‐enolpyruvylshikimate‐3‐phosphate synthase (CP4‐EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4‐EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4‐EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4‐EPSPS detection.

Thaulin-1: The first antimicrobial peptide isolated from the skin of a Patagonian frog Pleurodema thaul (Anura: Leptodactylidae: Leiuperinae) with activity against Escherichia coli

Patagonias biodiversity has been explored from many points of view, however, skin secretions of native amphibians have not been evaluated for antimicrobial peptide research until now. In this sense, Pleurodema thaul is the first amphibian specie to be studied from this large region of South America. Analysis of cDNA-encoding peptide in skin samples allowed identification of four new antimicrobial peptides. The predicted mature peptides were synthesized and all of them showed weak or null antimicrobial activity against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli with the exception of thaulin-1, a cationic 26-residue linear, amphipathic, Gly- and Leu-rich peptide with moderate antimicrobial activity against E. coli (MIC of 24.7μM). AFM and SPR studies suggested a preferential interaction between these peptides and bacterial membranes. Cytotoxicity assays showed that thaulin peptides had minimal effects at MIC concentrations towards human and animal cells. These are the first peptides described for amphibians of the Pleurodema genus. These findings highlight the potential of the Patagonian regions unexplored biodiversity as a source for new molecule discovery.

Isolation of Trypsin from Bovine Pancreas Using Immobilized Benzamidine and Peptide CTPR Ligands in Expanded Beds

Abstract Peptide CTPR and p‐amino benzamidine (PAB) immobilized on Streamline™ were utilized as the chromatographic matrices for trypsin purification from bovine pancreas. By using a clarified pancreas extract, maximum capacity for CTPR‐Streamline was 47.4 mg/mL and for PAB‐Streamline 78.9 mg/mL while Kd values were 0.39 and 0.38 respectively. Dynamic capacity was 23.0 and 46.0 mg/mL for CTPR‐ and PAB‐Streamline respectively. When the purification process was applied to unclarified pancreas extract in the expanded‐bed adsorption mode, 80% trypsin recovery with a purification factor of 18.7 was achieved. Cationic and anionic trypsin obtained from the affinity column were separated by ion‐exchange chromatography.

Structure and function of a novel antioxidant peptide from the skin of tropical frogs

ABSTRACT The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin‐I, has a molecular weight [M+H]+ = 1543.69 Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin‐I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin‐I has a novel primary sequence with low similarity compared with previously described amphibians AOPs. Antioxidin‐I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin‐I presented a low cytotoxicity and suppressed menadione‐induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia‐induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide. Graphical abstract Figure. No caption available. HighlightsAntioxidin‐I is a new antioxidant peptide isolated from the skin tropical frogs.The bioactive peptide presented very low cytotoxicity against mammalian cells.It was able to avoid redox imbalance in oxidative challenged cells.Antioxidin‐I had the capacity to suppress ROS levels in hypoxia‐exposed microglia.Results support the application of the peptide for neuroprotection.