1,721,086 research outputs found
Isoelectric focusing in immobilized pH gradients: recent analytical and preparative developments
No full textIsoelectric focusing in immobilized pH gradients (IPG), covering both analytical and preparative aspects, is here reviewed. An extensive introduction covers the development of the technique from its inception in 1982 to present day methodology, with particular emphasis on the development of computer programs able to calculate and optimize linear and nonlinear pH gradients, spanning as much as 9 pH units, from a mixture of as many as 10 different buffering ions and titrants. The unique resolving power of IPGs is illustrated with the resolution of fetal globin chains differing by an Ala/Gly substitution in residue 75, this bringing about a minute difference in pI value of only 0.001 pH units. IPG runs, performed under denaturing conditions, allow an excellent correlation between experimental and theoretical protein pIs, to the extent that outliers were found to be polypeptide chains which had undergone post-synthetic modifications. The IPG methodology allows easy interfacing with mass spectrometry, due to the fact that proteins eluted from an IPG gel are isoionic as well as isoelectric, and thus are not contaminated by any buffer ion. The review ends with an excursus on preparative aspects of IPGs: a novel apparatus, based on the principle of isoelectric, buffering membranes, allows pilot-scale purification of r-DNA proteins to extreme purity, with recovery in a liquid vein. Isoelectric membranes have a selectivity based on a continuous titration process, and thus act as isoelectric traps for individual protein species. This same preparative apparatus can be used as a novel immobilized enzyme reactor, with superior performance compared to conventional types of reactors
An isoelectrically trapped enzyme reactor operating under an electric field
No full textMembrane enzyme reactors constitute an attempt at integrating catalytic conversion, product separation and/or concentration and catalyst recovery into a single operation. Whereas conventional membrane reactors confine an enzyme, in a free form, to one side of a membrane by size exclusion, electrostatic repulsion, or physical or chemical immobilization onto an intermediate support (gel, liposome), the membrane reactor here described is shown to operate under an entirely new principle: enzyme confinement into an isoelectric trap located in a multicompartment electrolyzer operating in an electric field. Two isoelectric membranes, having pI values encompassing both the enzyme pI and the pH of its optimum of activity, act by continuously titrating the enzyme trapped inside, thus preventing it from escaping the reaction chamber. Charged products generated by the enzyme catalysis are continuously electrophoretically transported away from the reaction chamber and collected into other chambers stacked either towards the cathodic or anodic sides. In a urease reactor, ammonia is continuously harvested towards the cathode, thus allowing >95% substrate consumption with maintenance of enzyme integrity over much longer time periods than in a batch reactor. In a trypsin reactor, casein is digested and biologically active peptides are continuously harvested in a pure form into appropriate isoelectric traps. In a third example, pure D-phenylglycine is produced from a racemate mixture, via an acylation reaction onto a cosubstrate (the ester methyl-4-hydroxyphenyl acetate), brought about by the enzyme penicillin G acylase
Rational selection of peptide epitope templates for proteins imprinting
No full textBackground
The main disadvantage in the use of proteins as templates in protein imprinting is their 3D-structure, complex and flexible, that unfolds easily. Decreasing the complexity of the protein template seems the best strategy, as demostrated by “epitope imprinting”. Yet, in all current protein MIP approaches, internal epitopes were not accessible and thus, have never been considered as templates, bringing to the loss of a huge number of potential epitope-candidates. Furthermore, in many cases conserved epitopes of diagnostic interest are located at the core of the protein, where these are not exposed to the pressure of the immune system.
Objective
Here we propose a rational method for the selection of epitopes for protein imprinting that enables targeting all epitopes, either exposed or internal. Named fingerprint imprinted polymers (FIP), in analogy with the fingerprinting analysis (the use of the constituent peptides to identify a protein).
Methods
The FIP method is based on the following steps for identifying a peptide candidate for imprinting: (I) in silico cleavage of the protein sequence by various cleaving agents; (II) selection of the resulting peptides based on length and hydrophylicity ; (III) screening of each peptide candidate against the entire protein sequences databank (e.g. UniProtKB) to eliminate candidate sequences that are not unique enough. A probability score of uniqueness is associated to the each peptide candidate, allowing the selection of unique sequences as templates.
Results
To proof the principle, NT-proBNP maker of cardiovascular risk, was chosen. The in silico analysis of NT-proBNP sequence allowed to individuate two peptide candidates, next used as templates for the preparation of NT-pro-BNP specific FIPs and tested for their ability to bind the NT-proBNP peptides in complex samples. Results indicated remarkable imprinting factor (IF 10), binding capacity of 0.5-2 mg/g, in line with many affinity materials, ability to rebind the 40% of template in a complex sample, composed of the whole digests of NT-proBNP.
Conclusions
Results supported the validity of the method here proposed. Key point is the fragmentation of the protein into peptides. The advantage is the ability to circumvent the issue of the folding of the protein
Isoelectric focusing in immobilized pH gradients: an update.
No full textThe latest trends on isoelectric focusing (IEF) in immobilized pH gradients (IPG) are here reviewed. The major advances on IPG technologies have been made when interfacing this technique with sodium dodecyl sulfate-polyacrylamide gel electrophoresis to produce two-dimensional (2-D) maps. Previous 2-D maps were routinely performed using conventional IEF as a first dimension, which typically resulted in poor reproducibility of spot position. With IPGs, correlation between experimental and calculated protein pI values is as good as +0.01 to 0.02 pH units. A new software has also been released, permitting easy calculation and optimization of linear, concave and convex exponential gradients, even in very complex recipes utilizing all ten Immobiline chemicals. It has also been proven that IPGs can be interfaced with mass spectrometry, thus obtaining a novel 2-D map with the best of pI measurements in the first dimension coupled with the best of mass determination in the second dimension. Recently, it has been shown that IPGs can be exploited to charter forbidden grounds, with the creation of non-linear pH gradients covering the extreme alkaline pH 10-12 gradient. In such basic regions, excellent steady-state patterns of histones and subtilisin mutants have been reported. Different families of histones could be mapped not only in this pH 10-12 interval, but also in 2-D maps exploiting this very alkaline gradient in the first dimension. Although the IPG technique is now a trouble-free, user-friendly technique, some annoying artefacts, producing severe protein smears and precipitation, were very recently reported, but found to be linked to some commercial Immobiline preparations containing up to 5% oligomers. Better quality control on the part of the company producing such chemicals should eliminate even this last source of troubles
Molecularly imprinted polymers by epitope imprinting: a journey from molecular interactions to the available bioinformatics resources to scout for epitope templates
Full text linkThe molecular imprinting of proteins is the process of forming biomimetics with entailed protein-recognition by means of a template-assisted synthesis. Protein-imprinted polymers (pMIPs) have been successfully employed in separations, assays, sensors, and imaging. From a technical point of view, imprinting a protein is both costly, for protein expression and purification, and challenging, for the preservation of the protein's structural properties. In fact, the imprinting process needs to guarantee the preservation of the same protein three-dimensional conformation that later would be recognized. So far, the captivating idea to imprint just a portion of the protein, i.e., an epitope, instead of the whole, proved successful, offering reduced costs, compatibility with many synthetic conditions (solvents, pH, temperatures), and fine-tuning of the peptide sequence so to target specific physiological and functional conditions of the protein, such as post-translational modifications. Here, protein-protein interactions and the biochemical features of the epitopes are inspected, deriving lessons to prepare more effective pMIPs. Epitopes are categorized in linear or structured, immunogenic or not, located at the protein's surface or buried in its core and the imprinting strategies are discussed. Moreover, attention is given to freely available online bioinformatics resources that might offer key tools to gain further rationale amid the selection process of suitable epitopes templates
Fractionation of carrier ampholytes in multicompartment electrolyzers with isoelectric membranes
No full textMulticompartment electrolyzers with isoelectric membranes can be successfully utilized for the preparation of carrier ampholytes of narrow pH ranges from commercially available wider pH intervals. An example is given on the preparation of a 0.6 pH unit (pH 6.7-7.3) range from a standard pH 6-8 interval. A 10% Ampholine solution is focused in an electrolyzer equipped with the following isoelectric membranes: pI 6.0, 6.7, 7.3 and 8.0. The narrow pH 6.7-7.3 cut can be efficiently utilized for base-line separation and quantitation of hemoglobin A from its glycated form, Hb A,,. This analysis is important for the screening and follow up of diabetic patients. The advantage of multicompartment electrolyzers are: (i) precision in the preparation of narrow pH cuts, due to the presence of membranes with defined pr values; (ii) ability to perform in both small- and large-scale operations and (iii) absence of contaminants leaching from granulated supports, as typical of some previous techniques
Molecular imprinting using biopolymers as building Blocks: Sustainable and biocompatible metamaterials for smart recognition and selective biointerfaces
Full text linkThe present review provides a comprehensive analysis of the development and application of molecularly imprinted polymers (MIPs) derived from natural biopolymers, covering literature from the early 1980s onward. The discussion is organized based on the chemical nature of the biopolymers utilized, covering glucans, chitosan, alginates, proteins, and nucleic acids. Each section offers a description of the respective biopolymer, reports synthetic approaches and applications, and attempt a critical evaluation of its effectiveness in molecular imprinting. The use of biopolymers in MIP technology is a promising approach for producing highly selective and sustainable recognition systems. The review underscores the potential of biopolymer-based MIPs in advancing molecular imprinting technology and their impactful contributions to future applications
Production of D-Phenylglycine from racemic (D,L)-Phenylglycine via isoelectrically trapped penicillin G acylase
No full textPenicillin G acylase (PGA) is exploited for producing pure D-phenylglycine from a racemate mixture, via an acylation reaction onto a cosubstrate, the ester methyl-4-hydroxyphenyl acetate. The reaction, when carried in a batch, is severely hampered by the reverse process, by which the product, 4-hydroxyphenylacetyl-(L)phenyl glycine, upon consumption of L-phenylglycine, is converted by the enzyme back into free substrate and 4-hydroxyphenyl acetic acid via lysis of the amido bond. To prevent this noxious reaction, a multicompartment electrolyzer with isoelectric membranes (MIER) is used as enzyme reactor, operating in an electric field. PGA is trapped between pI 5.5 and pI 10.5 membranes, together with an amphoteric, isoelectric buffer (lysine). As the 4-hydroxyphenylacetyl-(L)phenyl glycine product is formed, it vacates the reaction chamber by electrophoretic transport and is collected close to the anode, in a chamber delimited by pI 2.5 and 4.0 membranes. The same fate occurs to the free acid 4-hydroxyphenyl acetic acid, formed upon spontaneous (and enzyme-driven) hydrolysis of the methyl ester in the reaction chamber. These combined processes leave behind, in the enzyme reaction chamber, the desired product, pure D-phenylglycine. The advantages of the MIER reactor over batch operations: the consumption of the L-form in the racemate is driven to completion and the enzyme is kept in a highly stable form, maintaining 100% activity after one day of operation, during which time the PGA enzyme, in the batch reactor, has already lost >75% catalytic activity
Capillary isoelectric focusing and isoelectric buffers: an evolving scenario.
No full textThe present review offers a new look at capillary isoelectric focusing (cIEF) by centering on the most troublesome aspects of the technique, namely: 1) how to modulate the slope of the pH gradient, for increasing resolution (equivalent to pH gradient engineering, as easily available in immobilized pH gradients); and 2) how to keep proteins in solution at (and in the proximity of) the pl value. A simple solution is offered in the first case: addition, to the standard 2-pH-units interval, of separators or spacers, i.e., of amphoteric molecules (either single or in combination) able to locally flatten the pH and increment resolution. Examples of the separation of fetal and glycated hemoglobins are provided. In the second case, a unique solubilization power (while maintaining full protein integrity and enzyme activity) is obtained if class I solubilizers are used. They consist of mixtures of sugars (e.g., sucrose and sorbitol) at ca. 1 M concentration, with zwitterions (up to 1 M) such as the class of nondetergent sulfobetaines, but also taurine and some of the Good's buffers (e.g., CAPS). In these solvents, the protein exists in a state of superhydration and its solubility is greatly augmented. The review ends with an excursus on the use of isoelectric buffers in zone electrophoretic separations. Such isoelectric buffers offer unique advantages: They permit very-high-voltage gradients (up to 1000 V/cm) and thus minimize analysis times (down to a few min in 30-35 cm long capillaries). This results in a marked increase in resolution, due to minimal diffusion-driven peak spreading. Such buffers are finding unique applications for generating peptide maps of tryptic digests of proteins and also in the analysis of oligonucleotides
Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field
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