Reviews in Molecular Biotechnology最新文献

DNA chips are an emerging technology for parallel detection of DNA molecules, with applications ranging from medicine to environmental monitoring. The typical set-up includes fluorescence labeling for detection of binding events on the chip surface. Here another labeling technique based on gold nanoparticles is presented. These labels are much more stable, and their optical signal is less influenced by the environment. The specificity of gold-labeled DNA probes and the ease of detection using optical reflection or transmission is demonstrated. In conclusion, gold-labeling is a promising candidate for more robust and reliable DNA-chip detection.

The efficiency of existing combinatorial biological library methods has been moderate in terms of the success rates, the affinities of the ligands selected and the time and effort involved in trying to optimize the initial leads. Although mimicking natural evolution, existing strategies take little notice of the importance of recombination within a selected population to generate increased diversity. We present an overview of our recent progress which has resulted in the successful development of such a strategy, which we designate cosmix-plexing^®. We incorporate recombination as a central feature in obtaining high success rates and high affinities, even for short monomer peptides, in a very short time. The method uses type II restriction enzymes to re-assort small hypervariable DNA cassettes from an intermediate pre-selected population (e.g. from a phagemid display library), while maintaining the original open-reading frame. Since, in the naive library, each cassette contains all possible combinations of the polypeptide sequences it encodes, much longer regions can be optimized than was possible with methods which depend on a simple selection from the naive library. Short peptides can now be rapidly selected, which exhibit the same, or higher, specificity and affinity for a defined target molecule, than (say) an antibody or even the natural ligand.

The development of soluble receptor proteins that recognise given target molecules — ranging from small chemical compounds to macromolecular structures at a cell surface, for example — is of ever increasing importance in the life sciences and biotechnology. For the past century this area of application was dominated by antibodies, which were traditionally generated via immunisation of animals but have recently also become available by means of protein engineering methods. The so-called ‘anticalins’ offer an alternative type of ligand-binding proteins, which has been constructed on the basis of lipocalins as a scaffold. The central element of this protein architecture is a β-barrel structure of eight antiparallel strands, which supports four loops at its open end. These loops form the natural binding site of the lipocalins and can be reshaped in vitro by extensive amino acid replacement, thus creating novel binding specificities. The bilin-binding protein (BBP) was employed as a model system for the preparation of a random library with 16 selectively mutagenized residues. Using bacterial phagemid display and colony screening techniques, several lipocalin variants — termed anticalins — have been selected from this library, exhibiting binding activity for compounds like fluorescein or digoxigenin. Anticalins possess high affinity and specificity for their prescribed ligands as well as fast binding kinetics, so that their functional properties are similar to those of antibodies. Compared with them, they exhibit however several advantages, including a smaller size, composition of a single polypeptide chain, and a simple set of four hypervariable loops that can be easily manipulated at the genetic level. Apart from haptenic compounds as targets, anticalins should also be able to recognise macromolecular antigens, provided that the random library is accordingly designed. Hence, they should not only serve as valuable reagents for bioanalytical purposes, but may also have a potential in replacing antibodies for medical therapy.

The creation of diversity in populations of polypeptides has become an important tool in the derivation of polypeptides with useful characteristics. This requires efficient methods to create diversity coupled with methods to select polypeptides with desired properties. In this review we describe the use of in vivo recombination as a powerful way to generate diversity. The novel principles for the recombination process and several applications of this process for the creation of phage antibody libraries are described. The advantage and disadvantages are discussed and possible future exploitation presented.

The antigen-binding capacity of the paired variable domains of an antibody is well established. The observation that the isolated heavy chains of anti-hapten antibodies retain some antigen-binding capacity in the absence of light chains led to attempts to obtain an even smaller antigen-binding unit in a VH format. Unfortunately, the poor solubility, the reduced affinity for the antigen and the irreproducible outcome showed that additional protein engineering would be required to successfully generate single-domain antibody fragments. By serendipity, it was discovered that this engineering is already performed continuously in nature. Part of the humoral immune response of camels and llamas is based largely on heavy-chain antibodies where the light chain is totally absent. These unique antibody isotypes interact with the antigen by virtue of only one single variable domain, referred to as VHH. Despite the absence of the VH–VL combinatorial diversity, these heavy-chain antibodies exhibit a broad antigen-binding repertoire by enlarging their hypervariable regions. Methods are described to tap the VHH repertoire of an immunised dromedary or llama. These VHH libraries contain a high titre of intact antigen-specific binders that were matured in vivo. Synthetic libraries of a ‘camelised’ human VH, a mouse VH or a camelid VHH scaffold with a randomised CDR3 could constitute a valid alternative to immune libraries to retrieve useful single-domain antigen binders. The recombinant VHH that are selected from such libraries are well expressed, highly soluble in aqueous environments and very robust. Some in vivo matured VHH were also shown to be potent enzyme inhibitors, and the low complexity of the antigen-binding site is an asset in the design of peptide mimetics. Because of their smaller size and the above properties, the VHH clearly offer added-value over conventional antibody fragments. They are expected to open perspectives as enzyme inhibitors and intrabodies, as modular building units for multivalent or multifunctional constructs, or as immuno-adsorbents and detection units in biosensors.

This review describes various methods for the attachment of phospholipid bilayers to solid supports. The simplest approach involves vesicle unrolling onto a surface that has been previously modified with a continuous self-assembled monolayer (SAM). The choice of a suitable SAM can lead to the formation of attached bilayers that have the desired biomimetic properties and are suitable for studying transmembrane proteins. However, there are intrinsic problems associated with this approach if one is interested in studying ion transport phenomena. In particular, the relatively low resistance values found for such bilayers do not permit studies of single ion channels. For such studies to be carried out the background leakage through the lipid film must be greatly reduced. In an attempt to reduce the problems of leakage we have formed patterned SAMs in which a blocking, hydrophobic, layer covers 90% of the electrode surface. The remaining portion of the surface, which is hydrophilic, supports the formation of a bilayer. This approach has led to an improvement in the quality of the bilayers formed but has still not provided bilayers with sufficiently high specific resistances to study single ion channels. Finally, we describe new approaches based on the formation of bilayers suspended over small apertures. These ‘suspended’ bilayers are similar in structure to those used in black lipid membrane experiments and give rise to highly blocking bilayer membranes. Unfortunately, this approach requires the use of solvents to create the suspended bilayer and they are relatively fragile.

This review reports the significance of bilayer lipid membranes on a solid support (sBLM) for the construction of biosensors. The methods of formation of lipid membranes on different solid supports including different metals (silver, gold, stainless steel), agar and conducting polymers are presented. Several examples of the application of electrostriction and dielectric relaxation methods for the study of mechanical properties and dynamics of solid supported bilayers have been shown. We demonstrated that these methods are useful for determination of the binding of enzymes and antibodies to sBLM, for the study of hybridization of nucleic acids on membrane surfaces and for the study of physical properties of modified supported membranes.

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology ‘semifluid membrane’ has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.