Article 3 Program will be modified to include the additional project milestones: ? Construct expression clones for a majority of the 1261 candidate biomarkers specified in Polanski and Anderson (2006) manuscript and screen them for expression and solubility. ? Carry out scale-up production of milligram quantities of N 15 labeled and non-labeled proteins constituting a subset of these proteins as specified by Nd. ? Carry out additional expression and solubility screening on the 1261 candidate biomarkers using alternate vectors to increase the success rate for production of soluble protein products. ? Carry out preliminary experiments to determine the feasibility of producing phosphorylated proteins by treating proteins expressed in E. coli with kinases. The Director of the Clinical Proteomic Technologies for Cancer programs at the NCI will be responsible for oversight of this project from the NCI. Other approved NCI staff will be responsible for the management of the project. The Principal Investigator of this DOE project will be responsible for oversight of the project from ANL. ANL will provide proteins and peptides as a resource to support clinical proteomic technology development and assessment for cancer research. The objective of this agreement is to support the development of human proteins and peptides for use by the research community that will enable development of assays, and standard reagents. NCI will make these resources available to the research community to foster discovery research. The proteins and peptides to be produced from this project will be those that have been identified. as important factors in cancer biological processes. The NCI has major initiatives to support the development and assessment of novel protein detection technologies. These technologies are designed to standardize proteomic technologies, reference reagents and standards, measurement protocols, instrumentation, and sample acquisition, preparation and analysis platforms. A critical component needed to achieve the goals of these initiatives will be the development and validation of proteomic reagents including peptides/proteins and antibodies. DOE has established unique capabilities and expertise in the production, qualification, and characterization of peptides and proteins. This experience currently supports the production of proteins that are used to develop crystal structures for the resolution of their 3-dimensional structures. Such high quality, well characterized, and readily available proteins and peptides will be a key resource in advancing proteomic technologies and capabilities and will be essential for developing highly specific antibodies to proteins associated with cancer. The goal of the NCI protein production program is the production, characterization, and distribution of peptides and proteins for use in the development and validation of clinical cancer proteomic technology platforms. Protein targets will be selected based on scientific rationale that considers the biological relevance of the discovery of the targets in the cancer proteome. A candidate list of peptides and proteins for the proposed project will be selected by NCI in consultation with ANL scientists. DOE will support the expression of the proteins through a variety of strategies using E. coli expression systems that employ a target analysis system to select protein targets based on informatics-based selection or target categorization (e.g. size, localization signals).
