ペプチドと抗体のファージディスプレイ

4月28日 (月)

7:00 am - 5:45 pm レジストレーション開始

複雑なシステムにおける選択: 機能的に多様なライブラリの創造 
SELECTION IN COMPLEX SYSTEMS: CREATING FUNCTIONALLY DIVERSE LIBRARIES

8:30 am Chairperson’s Opening Remarks
K. Dane Wittrup, Ph.D., J.R. Mares Professor, Chemical Engineering & Bioengineering, Massachusetts Institute of Technology

8:40 am Methods to Select and Evolve Transmembrane Helices
William F. DeGrado, Ph.D., George W. Raiziss Professor, Biochemistry and Biophysics, University of Pennsylvania School of Medicine
While a variety of methods exist to design proteins that recognize water-soluble regions of target proteins, companion methods for targeting transmembrane (TM) regions have not yet been successful, hindered by the lack of structural information and less developed force fields. Here we describe a strategy for the computational design of CHAMP (computed helical anti-membrane protein) peptides that target TM helices in a sequence-specific manner. We illustrate the utility of this method by designing CHAMP peptides that specifically recognize the TM helix various integrins. We also discuss the interplay between computational, genetic, and chemical methods for improving the affinity of the initially designed peptides.

9:10 Chaperone-Assisted Crystallography: Overcoming Barriers to Solving Structures of Membrane Proteins and Functional RNAs
Anthony Kossiakoff, Ph.D., Professor and Chair, Biochemistry & Molecular Biology, University of Chicago 
Over the last decade there has been impressive progress in X-ray structure determination of biologically important macromolecules. However, structure determinations of membrane proteins and functional RNA molecules have lagged far behind for a number of technical reasons. We have developed a high-throughput Phage display screening pipeline that produces multiple high affinity binders to these difficult targets using three different scaffolds. Screening strategies can be employed that target either the extracellular or cytoplasmic portion of the membrane protein and can trap desired conformational states. As examples, the open and closed state of the full-length KcsA potassium channel and the P4P6 RNA molecule will be described.

9:40 Engineering Proteins for Structural Analyses Using Shotgun Proteolysis 
Daniel Christ, Ph.D., The Garvan Institute of Medical Research and Conjoint Senior Lecturer, Faculty of Medicine, The University of New South Wales
The majority of proteins in nature are multi-domain proteins and the identification of domain boundaries remains a persistent problem. This is particularly the case for structural studies of proteins, where unit boundaries frequently have to be optimized using iterative, experimental approaches. We have developed an alternative strategy based on random DNA fragmentation and proteolysis of the encoded polypeptides on phage. Our method (shotgun proteolysis) represents a high-throughput alternative to the classical limited proteolysis method for the isolation of stable components of multi-domain proteins. We present several applications of the technology, including its application to membrane protein fragments.

10:10 Grand Opening Coffee Break in the Exhibit Hall

11:10 Discovering Selective Kinase Inhibitors Utilizing Small Molecules Tethered to a Phage-Display Library
Indraneel Ghosh, Ph.D., Assistant Professor, Department of Chemistry, University of Arizona
Small molecule protein kinase inhibitors are often promiscuous as they target the highly homologous ATP binding pocket. To discover selective kinase inhibitors we have developed a new fragment-based ligand selection methodology that allows for the discovery of protein surface-targeted peptides steered by an ATP active site binding warhead. This approach couples high-affinity but promiscuous small-molecule inhibitors to the large chemical space encompassed by biological libraries to afford highly potent and selective kinase inhibitors.

11:40 Co-Crystallization with Antibody Fragments Generated by Phage Display 
Syed Mir, Ph.D., Lecturer Clinical Biochemistry, Kashmir University and Member, Molecular Biology, Max-Planck Institute of Biophysics
Antibody mediated co-crystallization of membrane proteins is a powerful approach to obtain high quality crystals for X-ray diffraction studies. The potential of the technique is limited by the low availability of suitable antibody fragments. We have generated high-affinity binders from immune-avain phage-display antibody library, and showed their suitability for co-crystallization of membrane protein targets.

12:10pm Phage-Displayed Selections and Screens with Terpene Synthases 
Gregory Weiss, Ph.D., Associate Professor, Departments of Chemistry, Molecular Biology, and Biochemistry, University of California, Irvine
Terpenoids include structurally diverse antibiotics, chemotherapeutic agents, flavorings, and fragrances. Engineering of terpene synthases for the development of enzymes with new catalytic activities and product specificities represents a long sought goal. We report computational design, expression, and evaluation, in high and low throughput phage-displayed assays, a thermostable mutant of tobacco 5-epi-aristolochene synthase (TEAS) for the catalysis of carbocation cyclization reactions at elevated temperatures.

12:40 pm Presentation I Fab Screening Using Kinetic Arrays Sponsored by Michael Cicirelli, Ph.D., Director, Array Applications, Plexera Biosciences 1:10 Presentation II (Sponsorship Available)

1:40 Break

選択とスクリーニングの向上
IMPROVED SELECTION AND SCREENING

2:00 Chairperson’s Remarks 
Andrew M. Bradbury, M.B. B.S., Ph.D., Staff Scientist, Biosciences, Los Alamos National Laboratory

KEYNOTE PRESENTATION
2:05 Synbodies: Beyond Antibodies?
Stephen Albert Johnston, Ph.D., Director, Center for Innovations in Medicine, Biodesign Institute, Arizona State University
In spite of their evolutionary constraints, antibodies are still the gold standard for binding agents. Yet for many proteomic applications antibodies are inadequate. Spurred by our interest in creating a platform for pre-symptomatic diagnosis, we have tried to invent a new form of antibodies with a broad use profile. I will report on how “synbodies” are made and work.

 

2:35 Using Helper Plasmids in Phage Antibody Library Selection
Andrew M. Bradbury, M.B. B.S., Ph.D.
We recently described a method to avoid the use of helper phage in phage display, by using bacterial packaging cell lines containing helper plasmids. In this talk the application of helper plasmids to selection from phage anti-body libraries will be described.

3:05 Refreshment Break in the Exhibit Hall

3:45 Engineering Aglycosylated Antibodies with Effector Functions
George Georgiou, Ph.D., Department of Chemistry and Biochemistry, University of Texas, Austin
We will present an integrated technology for the discovery of IgG antibodies in E.coli including methods to screen very large libraries for the isolation of high-affinity binders. The engineering of aglycosylated Fc domains to pro-vide effector functions (i.e. FcR and C1q) will be described.

4:15 FACS-Proteomics: Novel Particle Arrays for Parallel Use of Up to 1400 Antibodies in a Sample
Fridtjof Lund-Johansen, M.D., Ph.D., Department of Hematology, Rikshospitalet University Hospital
A new platform for highly multiplexed protein analysis is presented. Particles with bound protein G and up to 700 different color codes are used to immobilize antibodies. Further multiplexing is achieved by variation of particle size. Particle mixtures are incubated with labeled proteins from cell lysates for analysis of protein pathways. The particles are analyzed by six color flow cytometry to detect hundreds of antibody targets simultaneously. Results from comprehensive analysis of the cell cycle machinery are presented.

4:45 Integration of Phage Display and Yeast Surface Display for Engineering Synthetic Protein Interaction Interfaces
Shohei Koide, Ph.D., Associate Professor, Department of Biochemistry and Molecular Biology, The University of Chicago
A particularly promising branch of directed evolution-based protein engineering is to create novel functional interfaces on alternative scaffolds using fully synthetic, rationally designed amino acid diversity. We have inte-grated phage display and yeast surface display for efficiently producing and sorting synthetic interface libraries. This presentation will highlight the complementary features of these molecular display methods, minimalist library design and novel binding proteins produced from the integrated pipeline.

5:15 Problem-Solving Break-Out Sessions 

6:00 – 6:45pm Networking Cocktail Reception in the Exhibit Hall