Protein database of Caenorhabditis elegans

Whole genome sequencing of the free-living nematode Caenorhabditis elegans is a prominent achievement in genomics and uncovers the existence of enormous known and unknown gene products. Characterization and linking of all gene products are the next challenging theme of biology. Genome-wide researches are already progressing on C. elegans and the fruits of these efforts are accessible through the internet. To link the sequence-function relationship, proteomic research has been applied to provide comprehensive information of the worm proteins. In addition to 2-dimensional gel electrophoresis for visualization of the proteome, recent advances in liquid chromatography (LC)-based technologies have allowed the large-scale analysis of proteins and are at cutting-edge of high-throughput analysis of focused proteome.     

Unstable proteins: how to subject them to chromatographic separations for purification procedures

The chromatographic separation of an unstable protein is often a challenge to the scientist working in the field of life sciences. Especially for the purification of sensitive enzymes, making use of conventional chromatographic techniques is difficult and frequently results in a complete loss of biological activity of the target protein. This report summarizes some general strategies that may help to keep unstable proteins in their native conformation during the rather harsh conditions of a purification procedure. In this context, a recently developed hollow fiber membrane module, suitable for performing on-line dialysis, is introduced and examples of its application to liquid column chromatography are given. Many innovative separation techniques, characterized by dramatic improvements in both performance and separation time, have recently been developed. Since the chromatographic separation of unstable proteins requires the use of modern state-of-the-art equipment and technology, emphasis is given to newly developed separation techniques such as expanded bed adsorption, perfusion chromatography, protein free flow electrophoresis and the use of tentacle gels. In addition, examples of recently published purifications of unstable proteins are discussed with respect to strategies ensuring the preservation of the native protein structure during chromatographic separation.     

Factors affecting protein interaction at sorbent interfaces

Interactions between surfaces and macromolecules are the fundamentals in separation and detection of diverse solutes. In this very brief review the central aspects of protein-surface interactions are discussed with the intention of identifying the important factors influencing such processes and placing them in relation to the established knowledge in this field. Some perspectives of new techniques related to scanning probe microscopy for studying interactions at the nanometer level are also discussed.     

Current trends in capillary isoelectric focusing of proteins

Isoelectric focusing (IEF) in thin capillaries is reviewed here. After an introduction on the genesis and chemistry of the carrier ampholyte buffers, different approaches to IEF are discussed and evaluated. The classical approach consists on IEF under conditions of suppressed electroosmotic (EOF) flow, usually obtained by covalently bonding hydrophilic polymers to the inner capillary wall. The other approach consists of IEF in dynamically (and partially) coated capillaries, so as to allow a reduced EOF flow to coexist with the IEF process, so that focusing and transport of the train of stacked bands occurs simultaneously. The various experimental parameters: focusing, elution and detection steps, pI measurements, as well as typical drawbacks, such as isoelectric precipitation are evaluated. The review ends with some examples of analytical separations, at the moment mostlyl limited to focusing of native hemoglobins (normal and point mutants). These separations are compared with those obtained by slab-gel IEF and in immobilized pH gradients.     

Stimuli-responsive separation of proteins using immobilized liposome chromatography

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k_s) of β-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k_s,max=5.57). The k_s values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k_s, therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (α-glucosidase, k_s,max=11.3 at pH 4; carbonic anhydrase from bovine, k_s,max=6.53 at pH 4). The difference in the height and/or the position of the peaks of the k_s–pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter.     

Protein recovery using gas–liquid dispersions

Two separation techniques, foam separation and colloidal gas aphrons (CGAs), both of which are based on gas–liquid dispersions, are compared as potential applications for protein recovery in downstream processing. The potential advantages of each method are described and the concentration and selectivity achieved with each method, for a range of proteins is discussed. The physical basis of foam separation is the preferential adsorption of surface active species at a gas–liquid interface, with surface inactive species remaining in bulk solution. When a solution containing surface active species is sparged with gas, a foam is produced at the surface: this foam can be collected, and upon collapse contains surface active species in a concentrated form. CGAs are microbubble dispersions (bubble diameters 10–100 μm) with high gas hold ups (>50%) and relatively high stability, which are formed by stirring a surfactant solution at speeds above a critical value (typically around 5000 rpm). It is expected that when proteins are brought into contact with aphrons, protein adsorbs to the surfactant through electrostatic and/or hydrophobic forces. The aphron phase can be separated easily from the bulk solution due to its buoyancy, thus allowing separation of protein in a concentrated form.     

Laser-induced fluorescence technique for DNA and proteins separated by capillary electrophoresis

Recent developments in capillary electrophoresis (CE) in conjunction with laser-induced fluorescence (LIF) using long-wavelength (maximum excitation wavelength>500 nm) dyes are reviewed. These dyes are particularly of interest when conducting the analyses of biopolymers by CE-LIF using He-Ne lasers. These systems are benefited from low background, low costs, easy maintenance, and compactness. Derivatizations of DNA and proteins with fluorescent or nonfluorescent chemicals can be carried out prior to, during, or after separations. With the advantages of sensitivity, rapidity, and high efficiency, the applications of CE-LIF to the analysis of polymerase chain reaction products, DNA sequencing, trace analysis of proteins, and single cell analysis have been presented.     

Separation used for purification of recombinant proteins

The purification of molecules from recombinant cells may be strongly influenced by the molecular biology of gene isolation and expression. At the beginning of the process there may be a demand for information on the minute amounts of proteins and thus for ever increasingly sensitive techniques. Purification of recombinant proteins can differ from conventional purifications in several ways, depending on the solubility of the protein, occurrence in inclusion bodies, creation of fusion proteins with tags that enable simpler purification. Sometimes a (re)naturation step is required to get a bioactive protein. On the other hand, the techniques used in separation are essentially the same as for purification from the natural source and environment.     

Lipoproteins: comparison of different separation strategies

This review describes two chromatographic techniques for the separation of three main classes of lipoproteins (HDLs, LDLs and VLDLs) from human serum: hydroxyapatite chromatography and counter-current chromatography. The HDLs, LDLs and VLDLs were purified by the combined use of the two chromatographic techniques without prior ultracentrifugation.     

Chromatography on cells and biomolecular assemblies

Red cells, biomembrane vesicles, proteoliposomes and liposomes non-covalently immobilized in gel particles or beads have been used as stationary phases for biomembrane affinity analyses and ion-exchange chromatographic separation. Lipid monolayers coupled to silica beads have been utilized for membrane protein purification in detergent solution and plant cell walls for group separation of macromolecules according to size and charge. Further methodological studies are essential to implement general practical application.     

Proteomics: general strategies and application to nutritionally relevant proteins

Proteomics as a subset of applied genomics technologies will be a key area of biology during the first decade or two of the new Millennium, and that it will have major impact, both directly and indirectly, on nutritional science. The aim of this review is to summarize information about general strategies of proteome and its application to important food proteins (plant, animal, and microbial). Methods are also described for protein separation, identification and determination. This article covers papers published within the last decade.     

Capillary electrophoresis procedures for serum protein analysis: comparison with established techniques

Methods using automated capillary electrophoresis (CE) instrumentation are available for serum protein electrophoresis with monoclonal band quantitation, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis separations. The advantages of CE over previous gel methods relate to the time and labour saved by the automated instrumentation. High pI monoclonal bands and cryoglobulin specimens can be successfully analysed by CE. However, if the CE application uses a standard company supplied kit, then the cost savings are often negated by the high cost of the kit. Improvements such as the inclusion of both a UV-Vis as well as a fluorescence detector as standard within the one commercial instrument, the production of coated IEF capillaries with a useful life of at least 100 samples, and the introduction of a capillary array into all commercial instrumentation would ensure greater use of CE within both the clinical and other protein laboratories.     

Recent advances in chromatographic and electrophoretic methods for the study of drug-protein interactions

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines various chromatographic and electrophoretic methods that have been developed to study such interactions. An overview of each technique is presented along with a discussion of its strengths, weaknesses and potential applications. Formats that are discussed include the use of both soluble and immobilized drugs or proteins, and approaches based on zonal elution, frontal analysis or vacancy peak measurements. Furthermore, examples are provided that illustrate the use of these methods in determining the overall extent of drug-protein binding, in examining the displacement of a drug by other agents and in measuring the equilibrium or rate constants for drug-protein interactions. Examples are also given demonstrating how the same methods, particularly when used in high-performance liquid chromatography or capillary electrophoresis systems, can be employed as rapid screening tools for investigating the binding of different forms of a chiral drug to a protein or the binding of different proteins and peptides to a given pharmaceutical agent.     

Structure and function of bovine growth hormone bovine growth hormone as an experimental model for studies of protein-protein interactions

Growth hormone (GH) is a polipeptide that controls the differentiation, growth and metabolism of many cell types, and is secreted from the hypophysis of all vertebrate species tested so far. Despite the overlapping evolutionary, structural, immunological and biological properties, it is well-known that GHs from distinct mammalian species have significant species-specific characteristics. The main purpose of this review is to highlight bovine GH (bGH) structural features related to its species-specific properties. Novel interest in bGH is also aroused by the advent of biotechnological methods for production of recombinant proteins. In fact recombinant bGH will have a great importance in veterinary medicine research and as a ‘high tech’ drug that needs to be monitored in zootechnical productions.     

Protein separation using membrane-encapsulated soluble ligand conjugates.

A new approach for isolating and recovering biological macromolecules using membrane-encapsulated soluble ligand conjugates was investigated. Membrane-encapsulated solid adsorbents have been successfully developed and employed in our laboratory to isolate and purify proteins and enzymes directly from culture broths. This new concept also makes it possible to use soluble ligand conjugates instead of solid adsorbents inside membrane capsules. In this work, model membrane-encapsulated soluble and insoluble ligands comprising Blue Dextran and Blue Sepharose entrapped within calcium alginate membranes were studied to compare adsorption characteristics such as capacities and rates. Experimental results suggest that membrane-encapsulated soluble ligands may be expected to result in higher overall adsorption capacity compared to membrane-encapsulated solid adsorbents with comparable adsorption rates.     

Practical application of aqueous two-phase partition to process development for the recovery of biological products

The practical application of aqueous two-phase systems (ATPS) to process development has been exploited for several years for the recovery of biological products. Unfortunately, this has not resulted in an extensive presence of the technique in commercial processes. Some of the main identified reasons for such situation involve the full understanding of the mechanism governing phase formation and the behaviour of solute partitioning in ATPS processes, the cost of phase forming polymers and the necessary extended time to understand the technique for process development. In this review paper, some of the practical disadvantages attributed to ATPS are addressed. The practical approach exploited to design ATPS processes, the application to achieve process integration, the increasing use for the recovery of high-value products and the recent development of alternative low cost ATPS, are discussed. It is proposed that the potential trend in the application of ATPS processes for the recovery of biological products will involve the recovery of high-value bio-particulate products with medical applications. This proposed trend in the application of ATPS will address the urgent need to rapidly and economically bring new biopharmaceutical products to market using scaleable and efficient bioprocess technology.     

Protein analysis of an individual Caenorhabditis elegans single-cell embryo by capillary electrophoresis

We present a simple one-dimensional electrophoretic map of the expressed proteins in a Caenorhabditis elegans embryo. The embryo was taken from an adult nematode, injected into a 50-μm I.D. capillary, and lysed. The proteins were fluorescently labeled and then separated by capillary electrophoresis and detected by laser-induced fluorescence. Over 20 components were resolved in the 22-min separation. The dynamic range was outstanding for this separation, noise in the baseline was less than 0.01% the amplitude of the largest component.