Phage antibodies as tools for proteomics

1. Phage antibodies as tools for proteomics Molecular & Microbial Ecology Laboratory, School of Earth and Environmental Sciences 황청연

2. Contents Ⅰ. Approaches to proteomics Ⅱ. Construction and application of phage antibody libraries Ⅲ. Phage antibodies in functional genomics Ⅳ. Phage antibodies in proteomics

3. Ⅰ. Approaches to proteomics • Two-dimensional gel electrophoresis & mass spectrometry - the resulting databases are heavily biased towards abundant proteins • Protein-specific molecular probes - functionally important proteins of low abundance could be detected - depending on the generation of specific probes at a rate and a scale

4. Ⅰ. Approaches to proteomics • Phage display technology - a polypeptide (capable of performing a function, typically the specific binding to a target of interest) can be displayed on the phage surface by inserting the gene coding for the polypeptide in the phage genome phenotype (binding) pⅢ tip of phage genotype Fig 1. Phage displaying a binding protein (redrawn from Viti 1999)

5. Ⅰ. Approaches to proteomics • Antibody - one important functional unit of the immune system - produced by the B cells - It has been estimated that a human makes at least 1015 different antibody molecules, each of them specific for a different antigenic determinant (epitope) - specific, sensitive and quantitative detection of proteins in tissues or extracts - great potential for functional genomics and proteomics

6. Ⅱ. Construction and application of phage antibody libraries • Display of antibody fragments on bacteriophage - the favored format of antibody fragment is single-chain FV (scFV) antigen binding site VH Fab (50 kD) CH1 VL CL CH2 whole Ab (150 kD) CH3 FV (25 kD) scFV (27 kD) Fig 2. Schematic representation of different antibody formats (redrawn from Viti 1999)

7. Ⅱ. Construction and application of phage antibody libraries • Construction of antibody libraries - DNA sequences encoding VH and VL domains are amplified by PCR - VH and VL domains are paired randomly, increasing functional diversity in the library - The scFV sequences are amplified by PCR using primers incorporating restriction sites - cloning into the phagemid vector Fig 3. Display of Fab frag- ments on filamentous phage (Hoogenboom et al. 1998)

8. Ⅱ. Construction and application of phage antibody libraries - transformed into E. coli by electroporation - after rescue with helper phage, the random combinatorial library of antibodies is displayed on phage Fig 4. Antibody display library (Viti 1999)

9. Ⅱ. Construction and application of phage antibody libraries • Library size and diversity - Specificity and affinity are desirable qualities in any antibody - The larger the library, the greater the likelihood that it will contain an antibody of very high affinity having the desired specificity The potential diversity of antibody can be estimated as follows. (1) Diversity due to combinatorial integration: 300VK x 4JK = 1.2x103 and 200VHx15Dx4J=12x103 (2) Diversity due to junctional alternatives (alternative readings at junction due to differences in reading frame, and lengths of J and D segments): VK to JK~ 3 and VH to D to J~40 (3) Diversity due to N segment for heavy chain > 10 (4) Hence combined diversity for each chain: light chains=3x103 and heavy chains > 5x106 (5) Conclusion: diversity due to combinatorial association of heavy and light chains > 1010. Additional factor due to somatic mutation >1030 (Winter & Milstein 1991)

10. Ⅱ. Construction and application of phage antibody libraries • Selection of specific antibodies from a phage library Next panning cycle Antibody library on phage Antigen column Amplification Fig 5. Selection of a binding specificity from a phage display library (Viti 1999) Elute Wash

11. Ⅱ. Construction and application of phage antibody libraries • High-throughput selections - small volumes of liquid (100 μl) are required - multiwell microtiter plates - a different antigen may be coated onto each well • Phage antibodies as reagents - Phage antibodies have routinely been successful in ELISA on protein or peptide antigens ELISA on whole cells or subcellular fractions flow cytometry immunocytochemistry

12. Ⅲ. Phage antibodies in functional genomics • The ‘ProAb’TM approach

13. Ⅲ. Phage antibodies in functional genomics • Making sense of ESTs by bioinformatics - there are more than 106 expressed sequence tags (ESTs) in databases (http://www.ncbi.nlm.nih.gov/dbEST/index.html) - to focus on a known protein with interesting biological function (and, ideally, a known structure) and to search for family member • Synthesizing antigens - 15 amino acid residue antigenic peptides have been found suitable - the synthetic peptides are washed by precipitation - liquid chromatography and electrospray mass spectrometry (more than 85% pure will be used as antigens)

14. Ⅲ. Phage antibodies in functional genomics • High-throughput selection of phage antibodies to peptide antigens - the basic selection method is panning - selection, elution of bound phage, infection spreading, colony-picking, arraying clones into 96-well plates, and screening for peptide-specific antibodies by ELISA are run automatically on commercial robots

15. Ⅳ. Phage antibodies in proteomics • Conventional selection of phage antibodies to membrane proteins - such proteins are particularly difficult to purify to use as immunogens (i.e. 7-transmembrane chemokine receptors) - the use of unpurified mixtures of antigens - mixing a small amount of cells expressing the desired cell surface protein with a large number of cells that do not

16. Ⅳ. Phage antibodies in proteomics • Guided selection to improve membrane protein selections - utilize a receptor’s cognate ligand as the guide molecule - phage binding in the proximity of the guide molecule would thus isolated • ProxiMolTM method and applications - horseradish peroxidase (HRP) - phage antibody library - biotin tyramine, hydrogen peroxide - streptavidin-magnetic beads - guide molecule : it must have affinity either for the target protein itself or for a molecule in close proximity to the target, and must be capable of being associated with

17. Ⅳ. Phage antibodies in proteomics

18. Ⅳ. Phage antibodies in proteomics • ProxiMolTM method applications in proteomics research - use of antibody clones from ProAbTM ouput as ‘tags’ to guide new selections - The ProxiMolTM technique to identify new members of protein or protein/DNA complexes