Display technology
Introduction to display technology What is display technology? Display technology classically involves the screening of a large library of expressed proteins using an immobilized ligand to characterize or discover new protein-protein interactions. Characteristics of display technology The key to rapid discovery and characterization of protein-ligand interactions is the ability to couple the proteins being screened (phenotype) with the genetic information encoding them (genotype). In spite of the huge advances in protein sequencing methods in recent years, it is faster and more convenient if the genetic information of a ligand is isolated simultaneously with the protein of interest. This is the most important characteristic of display technology. In addition, a large library of proteins must be generated and displayed to the ligand of interest. The larger the library, the greater is the chance of finding a binding partner. The third important factor is that it must be possible to amplify this library without introducing a bias for a particular type of DNA fragment/protein to enable successive unbiased rounds of screening against a particular ligand. A further attractive feature of a display system is the ability to introduce errors randomly into the encoding DNA sequence to isolate mutant proteins with altered binding affinities or binding kinetics (SELEX technology; affinity maturation). Uses of display technology Discovery of new ligands Characterization of protein-ligand interactions Affinity maturation of protein-ligand interactions There are a number of popular display technologies based on display of proteins or protein fragments on the surface of biological entities, for examples, phage display, yeast display, which rely on the replication of the organism to amplify the library and introduce errors. As an alternative to these in vivo systems, there is also the possibility to carry out the whole process in a tube. In this case in vitro-generated transcripts are translated in cell extracts and reverse transcription (RT)-PCR is used to amplify the genetic information after the ligand-mediated isolation of mRNA-ribosome-protein complexes has taken place (ribosome display). Display technologies with MACS® Products In all of these systems, magnetic sorting with MACS Technology enables an efficient and rapid isolation of interacting proteins. Library proteins interacting with labeled, for example, biotinylated, ligands can be isolated using Streptavidin or Anti-Biotin MicroBeads as shown in figure 1. If the ligand is an antibody, μMACS Protein A or Proterin G MicroBeads can be used. For sorting of cell-surface expressed libraries - Anti-Biotin MicroBeads - Streptavidin MicroBeads For sorting of molecular and phage libraries - µMACS Protein A MicroBeads - µMACS Protein G MicroBeads - µMACS Streptavidin Kit Display technology and magnetic sorting Advantages of magnetic sorting Large scale screening - orders of magnitude more events can be sorted compared to FACS Rapid magnetic isolation - sorting in a matter of minutes instead of days Convenient - no trained operator required Effective - very low non-specific enrichment compared to conventional panning techniques due to highly efficient wash steps Phage display 1011 bacteriophage particles can be sorted in one μ Column3 to Identify and analyse features of surface proteins that interact with other proteins Isolate new ligands that bind to particular amino acid sequences Improve the affinity and specificity of the interaction between ligands and their target structures Starting protocol using a biotinylated target (for example protein) and μMACS™ Streptavidin MicroBeads, available at www.miltenyibiotec.com. Yeast display Yeast display: 1010 yeast cells can be sorted in one LS Column2,6 or using the autoMACS™ Separator6 for: Correct post-translational modification, processing and folding of mammalian proteins Inducible expression allowing a 1010-fold expansion of a library without biased selection Rapid characterization of binding affinities of interacting proteins Protocol developed by Prof. Dane Wittrup of the Massachussetts Institute of Technology1 Full protocol available at http://www.sysbio.org/dataresources/singlechain.stm and Chao et al.6 Ribosome display Ribosome display: 1013 ribosome-mRNA complexes can be sorted in one μ Column4,5 to Select large protein libraries in vitro Amplify isolated RNAs by RT-PCR Further information µMACS™ Streptavidin Kit—phage display [PDF; 53,2 KB]		Figure 1 General working scheme for screening a library for ligand-binding activity using display technology with MACS® Technology. Figure 2 Magnetic labeling of a yeast display library using MACS® Technology. (Adapted from Feldhaus et al. 2003 Nat. Biotech. 21: 163-170)