In collaboration with Projects Ib and Ic, Project Ib1c1 is to serve as the support source of biochemical expertise including initial investigative approaches to the isolation, solubilization, and purification of both cell surface bound and extracellular matrix elaborated moieties, determination of optimal methods of fragmentation to yield peptides expressing a functioning epitope and which are sufficiently small to permit the final aim, amino acid sequence determination. Within this framework, the specific aims of Project Ib1c1 are: 1) generation of defined proteolytic digest fragments of purified tenascin for epitope restriction analysis, as immunogen for production of second and later generation antibodies to defined epitopes of interest, and as source for further fragmentation; 2) sequence analysis of peptides retaining anti-tenascin antibody binding capacity to map eptiopes; 3) purification and characterization of cell surface and extracellular matrix associated protein antigens defined by Mabs C12, D12, and E9, and those Mabs generated vs medulloblastoma cell surface proteins, for potential biochemical analysis as for tenascin; 4) analysis, fragmentation, and sequencing of the variant forms of the EGFR expressed by gliomas as identified in project Ic and reactive with anti-EGFR Mabs produced in project Ib. The long term association of this laboratory with the characterization of tenascin, our ongoing analyses of the receptor recognition site of alpha2M as a model for molecular dissection, and the capacity of this facility for biochemical analysis through sequencing combine to provide a strong protein chemistry source for projects Ib and Ic.
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