Display Technologies: An Approach to Functional Genomics
Guest Editor: Reto Crameri
Alternative Bacteriophage Display Systems Pp. 121-133
Luisa
Castagnoli, Adriana Zucconi, Michele Quondam, Mario Rossi, Paola Vaccaro,
Shotgun Phage Display Cloning Pp. 135-143
Karin
Jacobsson and Lars Frykberg
The Powerful Combination of Phage Surface
Display of cDNA Libraries and
High Throughput Screening Pp. 145-155
Reto Crameri
and Rimantas Kodzius
Peptide Display in Functional Genomics Pp. 157-169
Igor Fisch
Surface Display on Gram Positive
Bacteria Pp. 171-184
Marianne
Hansson , Patrik Samuelson, Elin Gunneriusson and
Stefan Ståhl
The Baculovirus Expression System as a Tool
for Generating Diversity by
Viral Surface Display Pp.185-192
Reingard
Grabherr and Wolfgang Ernst
High-throughput Screening of Surface
Displayed Gene Products Pp. 193-205
Peptidomics: The Comprehensive Analysis of
Peptides in Complex Biological
Peter
Schulz-Knappe, Hans-Dieter Zucht, Gabriele Heine, Michael Jürgens, Rüdiger Hess
and
Michael
Schrader
[Back to
top] Alternative Bacteriophage Display Systems
Luisa
Castagnoli, Adriana Zucconi, Michele Quondam, Mario Rossi, Paola Vaccaro,
Simona
Panni, Serena Paoluzi, Elena Santonico, Luciana Dente and Gianni Cesareni
Filamentous
phage has been extensively used to implement various aspects of phage display
technology. The success of these organisms as vectors to present foreign
peptides and to link them to their coding sequences is a consequence of their
structural and biological characteristics. Some of these properties, however,
represent a limitation when one attempts to display proteins that cannot be
efficiently exported through the bacterial membrane or do not fold properly in
the periplasm. Thus, the desirability of developing alternative display systems
was recognised recently and led to the development of a different class of
display vectors that assemble their capsid in the cytoplasm and are released
via cell lysis. This review describes and compares the properties of these
alternative display systems.
[Back to
top] Shotgun Phage Display Cloning
Karin
Jacobsson and Lars Frykberg
Shotgun phage display cloning is a useful tool for studying interactions between bacterial and host proteins. Libraries are constructed by cloning randomly fragmented prokaryotic DNA into phagemid-vectors. Theoretically, these libraries will consist of phages that together display all proteins encoded by the bacterial genome. Selecting a gene III-based library, made from Staphylococcus aureus DNA, against IgG and fibronectin resulted in 20-40% positive clones after two pannings. Increasing the number of fusion proteins per phage particle by using gene VIII-based display, increased the frequency of correct clones to 75-100%.
[Back to
top] The Powerful
Combination of Phage Surface Display of cDNA Libraries and
High Throughput Screening
Phage surface display of cDNA libraries
facilitates cloning, expression and rapid selection of functional gene products
physically linked to their genetic information through gene product-ligand
interactions. Efficient screening technologies based on selective enrichment of
clones expressing desired gene products allows, within a short time, the
isolation of all ligand-specific clones that are present in a library. Manual
identification of clones by restriction analysis and random sequencing is
unlike to be successful for the isolation of gene products derived from rare
mRNA species resulting from selection of the libraries using polyvalent ligands
like serum from patients. Here we describe rapid handling of large numbers of
individual clones selected from molecular libraries displayed on phage surface
using the power of robotics-based high throughput screening. The potential of
the combination of cDNA-phage surface display, with selection for specific
interactions by functional screening and robotic technology is illustrated by
the isolation of more sequences potentially encoding IgE-binding proteins than
postulated from Western blot analyses using extracts derived from raw material
of complex allergenic sources. The subsequent application of functional
enrichment and robotics-based screening will facilitate the rapid generation of
information about the repertoire of protein structures involved in allergic
diseases.
[Back to
top] Peptide Display in Functional Genomics
The completion of the human genome project has opened novel scientific avenues in functional genomics, structural genomics and proteomics. These areas have a common goal: the identification of all the proteins acting and cross-talking in a single cell at a defined moment of its lifecycle. The expansion of these areas in bioscience has been facilitated by the rapid development of high throughput screening (HTS) methods which has, in turn, attracted the business community to make investments in this novel business segment of biotechnology. By using these HTS methods, the hope is that novel targets will be validated much more rapidly speeding up the development of novel drugs. Numerous techniques and tools have emerged over the past decade for the identification of small target-specific molecular ligands that exploit a common feature: the exploration of molecular diversity using combinatorial methods. While chemists developed new methods for rapidly and efficiently synthesising and screening large collections of small molecules, biologists used recombinant DNA techniques for selecting displayed repertoires. To this end, the discovery of new low molecular weight peptides is becoming increasingly important, not only as molecular tools for the understanding of protein-protein interactions but also for the generation of lead compounds.
[Back to
top] Surface Display on Gram Positive Bacteria
Marianne
Hansson , Patrik Samuelson, Elin Gunneriusson and
Stefan Ståhl
Heterologous surface display on Gram-positive bacteria was first described almost a decade ago and has since then developed into an active research area. Gram-positive bacterial surface display has today found a range of applications, in immunology, microbiology and biotechnology. Live bacterial vaccine delivery vehicles are being developed through the surface display of selected foreign antigens on the bacterial surfaces. In this field, "second generation" vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered Gram-positive bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other variants of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. This article explains the basis of Gram-positive bacterial surface display, and discusses current uses and possible future trends of this emerging technology..
[Back to
top] The Baculovirus Expression System as a Tool
for Generating Diversity by
Viral Surface Display
It has become a major goal of molecular biologists, biochemists, and immunologists to be able to modulate the structure of proteins, in order to increase their antigenicity, alter their biological properties and/or explore their function. Based on the concept of bacterial phage display, by which proteins are being selected and analyzed in conjunction with their genetic information, eukaryotic systems have been investigated for their use in generating biomolecular diversity. The advantage of posttranslational modification and the possible harbouring of structural complex proteins has lead scientists to include eukaryotic systems in the wide field of molecular design. The ideal expression vectors for surface display are eukaryotic viruses, that allow large gene insertions, efficiently present foreign proteins on the particle surface, are easy to propagate and, if possible, not pathogenic to humans. By inserting peptides into a native virus coat protein or by expressing foreign proteins as coat protein fusion proteins or linked to specific anchor domains it becomes possible to display polypeptides of interest on the surface of replicating particles. A variety of different strategies are currently under investigation in order to utilize the baculovirus insect cell expression system for efficient display on the surface of virus particles as well as on the surface of virally infected insect cells. Increasing the transfection efficiency, optimizing cloning procedures, and establishing applicable selection methods have lead to the development of a powerful tool for drug screening and ligand screening.
[Back to
top] High-throughput Screening of Surface
Displayed Gene Products
With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phagemid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries..
[Back to
top] Peptidomics: The Comprehensive Analysis of
Peptides in Complex Biological
Peter
Schulz-Knappe, Hans-Dieter Zucht, Gabriele Heine, Michael Jürgens, Rüdiger Hess
and
Michael Schrader
Progress in the sequencing of genomes has resulted in an increasing demand for a functional analysis of gene products in order to understand the underlying physiology. Proteomics has established itself as a highly valuable technology for producing functionally related data in an unparalleled fashion, but is methodologically restricted to the analysis of proteins with higher molecular masses (>10 kDa). The development of a technology which covers peptides with low molecular weight and small proteins (0.5 to 15 kDa) was necessary, since peptides, amongst them families of hormones, cytokines and growth factors, play a central role in many biological processes. To summarise the technologies used for this approach the term "peptidomics” is introduced. In this article, we present the rationale and first results of a novel, universal peptide display approach for the analysis and visualisation of peptides and small proteins from biological samples. Special attention is given to samples derived from extracellular fluids such as blood plasma and cerebrospinal fluid. Additionally, a high throughput identification procedure for the analysis of peptides in their native and processed molecular form is outlined.