Silvia A. Camperi

Immobilized metal ion affinity hollow-fibre membranes obtained by the direct grafting technique

Abstract An immobilized metal ion affinity hollow fibre was prepared by radiation-induced direct grafting of glycidylmethacrylate (GMA) on hydrophilized polyethylene membranes. The epoxy group was converted into an iminodiacetate by iminodiacetic acid treatment. The effect of the radiation dose, salt inhibitors, methanol and GMA concentration, on the grafting degree was studied. The degree of grafting was closely related to the GMA concentration. Salt inhibitors failed in the production of a differential effect on the grafting and homopolymerization processes. Between 30 and 35% of methanol, there is a maximum yield, and no grafting was obtained with a methanol concentration above 50%. Water flux dropped exponentially with the increase in the grafting degree. Scanning-electron microscopy showed that the graft branches are formed on the pores. The pectinesterase adsorption capacity of the membranes was of the same order as of the commercial chelating soft gels.

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.

Protein Adsorption onto Tentacle Cation-Exchange Hollow-Fiber Membranes

A sulfonic group (up to 200 μmol/mL) membrane was incorporated to epoxy‐activated microporous hollow fibers to obtain high‐capacity convective ion exchangers. The pure water flux through the membrane decreased exponentially with sulfonic group density and protein binding capacity increased accordingly. At sulfonic group density of 70 μmol/mL, the membrane lysozyme maximum binding capacity was 84 ± 9 mg/mL in comparison with its theoretical monolayer maximum binding capacity of 20 mg/mL, thus evidencing tentacle formation. After a cycle of adsorption in a 30 mM sodium phosphate buffer, pH 7.0, adsorbed lysozyme could be quantitatively recovered following elution with 0.5 M NaCl in the same buffer. Dynamic capacity for lysozyme was 67% of maximum binding, and this value did not change at space velocities ranging from 10 to 40 min−1 as shown by the superimposition of the corresponding breakthrough curves. A cartridge assembled with 21 fibers showed a dynamic‐to‐static capacity ratio for lysozyme of 0.60 with 1 mg/mL pure lysozyme solution, and 0.42 with a particulate feed composed of 1 mg/mL lysozyme and 0.1 mg/mL yeast.

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.

Direct lysozyme separation from egg white by dye membrane affinity chromatography

An affinity membrane from hydrophylised polyethylene hollow fibre as the support matrix was prepared. Red HE-3B was immobilised on the membrane and the adsorption behaviour of pure lysozyme solutions and homogenised egg white was investigated. Dye density (1.7 μmol ml−1) and maximum binding capacity (26 mg lysozyme ml−1) are comparable to those of commercial gel matrices. Dynamic binding capacity did not change when residence time was reduced from 3 to 1 min. A method for direct lysozyme separation from egg white was developed, with a productivity of 12 kg lysozyme m−3 h−1. The purity of the eluted lysozyme, as determined by HPLC, was 88%, with a recovery of 92%. Dynamic capacity was kept constant at 70% of the maximum binding capacity for at least 10 cycles through membrane regeneration with 0.1 M NaOH and 1 M NaCl. Functional properties of egg white, as judged by viscosity and foaming capacity measurements, did not change after the chromatographic lysozyme depletion. © 1999 Society of Chemical Industry

Preparation and characterisation of immobilised metal ion hollow-fibre polysulphone membranes. Their application in high-speed pectic enzyme fractionation

Abstract Chelating hollow-fibre membranes were prepared from epoxy-activated polysulphone microfiltration fibres by introducing iminodiacetic acid (IDA) groups in the presence of dimethyl sulphoxide. Fibres with 160, 350 and 620 μmol epoxy groups/ml provided ligand densities of 69, 134 and 203 μmol IDA/ml and pure water fluxes of 7.8, 5.8 and 0.42 cm/min, respectively. However, lysozyme capacity was close to 4 μmol/ml for all fibres. Adsorption isotherms for lysozyme and pectinesterase did not fit Langmuir-type curves and the existence of two types of ligand (A and B) with different accessibility to proteins was assumed. For pectinesterase, maximum capacities of 5100 and 2900 U/ml and dissociation constants of 25 and 316 U/ml were found, respectively, for ligands A and B. For lysozyme, maximum capacities were 2.9 and 0.9 μmol/ml and dissociation constants 5.0 and 102 μM, respectively, for said ligands. A cartridge assembled with IDA hollow fibres had a dynamic capacity for pectinesterase of 7509 U/ml. Productivity of this cartridge for pectic enzyme fractionation was 750 pectinesterase U/ml min, far higher than that obtained with a chelating soft gel (81 pectinesterase U/ml min).

Chromatographic characterization of immobilized metal ion hollow-fiber affinity membranes obtained by direct grafting

Abstract Iminodiacetic acid was immobilized onto membranes with different grafting degrees by reaction in phosphate buffer or water/dimethyl sulfoxide. Membranes subjected to conversion in water/dimethyl sulfoxide underwent greater conversion than those modified in phosphate buffer, despite their grafting degree. Copper saturation capacity increased consistently with the grafting degree and histidine saturation capacity was approximately half than that of copper. When working with proteins, membrane behavior was related to the molecular weight of the protein tested. Accessible sites for lysozyme decreased with the increase in the grafting degree and the rise in the conversion of epoxy groups in iminodiacetic groups in water/dimethyl sulfoxide while they remained practically unchanged when the conversion step was performed in phosphate buffer. When working with hemoglobin, this effect was the same but at lower capacities. For hollow fibres with 60 and 75% grafting, capacities were the same despite the conv...

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.

Study of variables involved in fungal pectic enzyme fractionation by immobilized metal ion affinity chromatography

Abstract The effects of the pH, composition and ionic strength of the chromatographic buffer and immobilized metal on the fractionation of a commercial pectic enzyme were studied using immobilized metal ion affinity chromatography. The best performance of the system was achieved with Cu 2+ as the immobilized metal, 20 m m sodium phosphate (pH 7·0 containing 100 m m NaCl) as the adsorption buffer and 100 m m sodium acetate (pH 3·0 containing 100 m m NaCl) as the elution buffer. Under these conditions, pectinesterase was fully retained by the chromatographic matrix while the fraction passing through — containing pectin lyase — allows clarification of fruit juice without methanol production. The retained pectinesterase and the polygalacturonase contained in the commercial pectic enzyme preparation can be eluted quantitatively and applied to other industrial uses. From equilibrium adsorption isotherms, the maximum capacity of Chelating Sepharose for pectinesterase under the above conditions was 2990±190 U/ml at 20°C and 2940±170 U/ml at 30°C, while K d values were 27·1±5·3 U/ml and 57·6±9·5 U/ml, respectively. From breakthrough curves, the optimum linear flow rate was 0·80 cm/min with a commercial preparation concentration at 100 mg/ml.

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.