With the recent completion of sequencing many bacterial and eukaryotic
genomes, we are entering a "post-genomics era". To add value to this
accomplishment, the scientific community’s attention is now directed at
determining the function of the thousands of gene products in each cell.
Traditionally, one valuable type of reagent that is widely used to probe cells and
learn when the protein product of a gene is synthesized, where it is localized, and
what it is associated within the cell is the antibody. However, it typically takes 2-3
months to generate rabbit or mouse antibodies to each individual protein, and
there is limited control by the investigator on the quality of the antibodies
generated by the immunized animals. To overcome the limitations of generating
antibodies and to meet the need for thousands of antibodies, we propose to use
high-throughput molecular biology, phage-display, and biochemical techniques to
isolate high-affinity and selective "designer affinity reagents". It is now possible to
engineer in bacteria the expression of small fragments of human antibodies or
engineered protein scaffolds that are capable of binding to almost any protein.
Our goal is to have the ability to generate affinity reagents, in two to four weeks
time, to any bacterial, viral, or eukaryotic protein.
Once we have isolated affinity reagents and ascertained their binding strength
and selectivity, we propose to employ them in several different manners. First,
we will utilize them to affinity purify the target proteins, and then identify
interacting proteins through mass spectrometry. By identifying the interacting
partners of each protein, we can collect data from which we can generate
hypotheses regarding cellular function. Second, we will express them inside cells
where they will have an opportunity to bind to their target and monitor or inhibit
activity. Demonstration of a "phenotype" will provide clues regarding the protein’s
cellular function. Third, we will test the ability of the antibodies to promote the
crystallization of proteins for x-ray diffraction studies. There have been a number
of published successes in promoting the crystallization of different membrane
proteins with antibody fragments and the DARPins protein scaffold. Fourth, we
will array the affinity reagents and measure the concentration of many proteins
simultaneously in cells, as they respond to stimuli or become diseased. Such
measurements will aid ongoing systems biology efforts.