The Expression and Molecular Biology (EMB) Core will facilitate the design and construction of vectors for high level expression of soluble target proteins and protein complexes for SBDR Program Project members and will stimulate synergy between members by streamlining access to critical protein, antibody and cDNA reagents. This will be accomplished through three service-related aims and a fourth aim that will provide technique development for optimizing expression of protein fragments and interacting domains.
Aim 1 is to use available technologies to optimize expression of soluble proteins, protein fragments, and protein complexes, concentrating on difficult-to-express proteins. Several strategies will be used to get highly expressed, soluble protein, including using various vectors and hosts that are part of the Gateway cloning system, dissecting large proteins into functional domains, and co-expressing interacting proteins and domains.
Aim 2 is to delineate and characterize selected protein domains for optimization of targets for crystallization. Limited proteolysis and mass spectrometry will be used to map protein-protein and protein-DNA interacting domains and to define potential heterogeneity that could interfere with crystallization. This Core will also assist in optimizing isolation of multi-protein complexes for biochemical and crystallographic analyses. Protein-protein and protein-DNA interactions will be measured by analytical gel filtration, surface plasmon, resonance, and spectrofluorometric techniques.
Aim 3 is to establish and maintain a Repository/Distribution Center of purified proteins, antibodies and cDNA clones associated with our collaborative research program, in order to maximize the resources of the program project. The EMB Core will be responsible for cataloging these reagents and for distributing them too requesting member laboratories will be available on a secure Web-based database.
Aim 4 is to develop high throughput protein biochemistry methods for optimizing expression of soluble protein fragments, interacting domains, and complexes. We will develop methods for producing expression libraries of nested deletion subclones with endpoints clustered near the predicted boundaries of a protein domain, coupled with screens or selections for protein fragments that are highly over-expressed in a soluble form. The EMB Core of SBDR will be comprised of two laboratories, one at LBNL and one at Harvard Medical School (HMS).
Aims 1 -3 will be implemented at LBNL and Aim 4 at the HMS site. As a centralized source of reagents, instrumentation, and expertise, this Core will greatly accelerate the pace of research for all five projects.
