- Chemical Biology and Medicinal Chemistry
- Discovering Enzyme Substrates and Functions
- Discovering Protein Ligands to Probe and Alter Function
- Discovering Enzyme Activators
- Analyzing Mechanisms of Drug Resistance via Chemical Biology
- Analyzing Enzyme Conformational Dynamics, Substrate Binding, and Catalysis
- Effective Drug Targeting of Pathogens via Medicinal Chemistry
- Computational Chemistry and Biology
- Physical Biology
- Protein and Cellular Engineering
- Monitoring enzyme activity and disease biomarkers
- Generating human proteome antibodies via phage display and directed evolution
- Globally analyzing and dissecting apoptosis
- Proximity tagging of protein-protein interactions
- Investigating cellular interactions in tissues
- Creating fluorescent probes targeting the genome and key bio-pathways
- De novo design of catalytic and membrane proteins
- Probing and modulating membrane proteins
- Chemical Biology and Medicinal Chemistry
Generating human proteome antibodies via phage display and directed evolution
Examples of our research, methods, and facilities include
UCSF Antibiome Center
The department is home to the UCSF Antibiome Center, a high-throughput robotic platform capable of generating recombinant antibodies of superior quality in a fraction of the time of traditional techniques by combining phage display with automated selection. The Center is one of three NIH-funded laboratories in North America comprising the Recombinant Antibody Network.
The Center’s initial charge is to provide the wider research community with high quality, open source, renewable antibodies selective for the approximately 1,500 human transcription factors—proteins that bind to DNA and regulate gene expression. They are to be used, in particular, for chromatin immunoprecipitation (ChIP) and immunofluorescence studies to determine where those proteins bind and which co-factors they recruit to produce their specific effects.
There are plans to expand this effort to include all cell surface and secreted proteins. A longer-term goal is produce these reagents for the entire human proteome, including for specific post-translational modifications, with potential uses for research, diagnostic assays, and therapeutic agents.
Antibody phage display is a technique by which the genes that encode the part of an antibody that binds antigens are inserted into the DNA of a bacteriophage (also known as a phage, a virus that replicates itself inside bacteria) or, at the Center here, a phagemid (a hybrid of phage and a plasmid, a DNA molecule that replicates inside bacteria).
These genes then express fragment antigen-binding (Fab) antibody fragments. These Fab proteins are displayed on the ends of the bacteriophage particles released by the bacteria. (With some additional engineering, Fabs can be made into full-size antibodies.)
In order to engineer antibodies that bind to proteins of interest selectively (high specificity) and tightly (high affinity), scientists here focus on the antigen binding sites at the tips of the Y-shaped immune proteins. Those sites, in turn, have six complementarity determining regions (CDRs) that precisely complement a given antigen’s shape. By substituting different combinations of amino acids at key locations in the CDRs, as well as altering the lengths of their loop-shaped structures, researchers generate recombinant libraries comprised of more than 10 billion different Fab molecules.
Automated selection / directed evolution
To select a recombinant antibody for a given protein, the Center’s researchers use an automated robotic platform to fish the best-binding Fab from that collection (library) of 10 billion-plus. The protein targets are labeled with biotin allowing them to be captured on streptavidin-coated paramagnetic beads (biotin and streptavidin form a strong non-covalent bond). Specialized robots transfer the immobilized proteins-on-beads into the Fab library mixture, allowing the specific antibody molecules to bind to them and be recovered.
During the initial stages of selection there is substantial non-specific binding to the protein, its co-factors, and tags. But iterative rounds of selection for binding to the antigen winnow the Fabs down to only those that bind the target with high affinity and specificity. The Center uses a combination of automated and manual validation assays to determine affinity and specificity of each of the Fab reagents prior to release for the general scientific community.
Our applications of such protein engineering include
Selecting recombinant antibodies for cancer-related proteases
Generating antibodies via phage display that selectively bind to the active forms of certain cell membrane-tethered proteases (matriptase, prostate specific antigen, urokinase-type plasminogen activator) that are dysregulated during various stages of cancer progression. Ongoing research is using immunofluorescence combined with the development of high-throughput multiplex microarrays to monitor the proteases’ activity for the diagnosis and staging of the disease as well as for evaluation of treatment efficacy.
Developing a strategy to make antibodies for phosphorylated peptides
More than 500 kinase enzymes add phosphate groups to up to 30 percent of all eukaryotic proteins, altering their function. This ubiquitous regulatory modification goes awry in many diseases, including cancers and neurodegenerative disorders. Antibodies are needed to detect these specific modifications in cells and tissues to facilitate understanding of signaling and as potential biomarkers.
Researchers here built on their knowledge of how proteins recognize and bind anions, like negatively charged phosphates. They sought out the most commonly employed structural motif—a binding pocket including three consecutive residues where multiple backbone amides form hydrogen bonds with anions—among a database of mAb structures. They found something similar in a mouse Fab and used it as a scaffold to build humanized phage display libraries.
First, they optimized a single Fab CDR’s loop structure (randomizing residues to complete the binding pocket, altering loop length to enlarge it) in order to bind the most common phosphorylated residues – serine, threonine, and tyrosine. Then, using these new Fab scaffolds, they altered other mAb regions (two additional CDRs) generating billions of different mAbs. Via rounds of competitive assays (directed evolution), researchers selected for mAbs that bind phosphorylated residues in context, e.g. a specific site on a protein. These selections led to the identification of 51 phospho-specific monoclonal antibodies.
Department scientists are now seeking to apply this approach to generating renewable antibodies to other common post-translational modifications.
Engineering conformation-selective antibodies
Virtually all enzymes are regulated in cells through conformational switching from on- to off-states. Antibodies not only recognize specific proteins, but also the specific conformational state of that protein. Department scientists have shown it is possible to deliberately isolate such conformationally selective antibodies by first locking the protein in their respective on- or off-states using known or discovered small molecule ligands that trap these states, or by using forms of the protein that are locked in these states naturally. They can then select antibodies for the protein state of interest and against the opposite state using in vitro phage display. This has allowed researchers here to identify antibodies that specifically activate or inhibit proteins by allosteric means.
In one study, this strategy was demonstrated with a protease that plays a key pro-inflammation role in the innate immune response (caspase-1) as a model target. Department scientists used allosteric and active site inhibitors to generate on- and off-state antigens. The resulting antibodies for those states were used to determine where the enzyme is active in cells.