The need for purified antigen, whether lipopolysaccharide (LPS) or protein, is often a roadblock for the efficient development of diagnostic methods or preventative measures targeting bacterial pathogens. The Antigen Production Core Laboratory (APCL) is designed as a user-directed facility for the generation of macromolecules useful in the study of enteric pathogens. Services rendered will include custom protein synthesis and the preparation of LPS. Expertise with the preparation of proteins prone to aggregation due to polymerizing properties or hydrophobic properties will be provided based upon extensive experience with the production of proteins (and LPS in some cases) from the Shigella, Salmonella, Yersinia, Pseudomonas and Burkholderia systems. An understanding of the structural, dynamic, and functional properties of targeted proteins by the Director and Co-Director of the APCL will also greatly facilitate efforts in producing the bacterial antigens requested by the individual investigators. Each protein to be prepared can present its own set of difficulties at any given step in its preparation, and overcoming such problems has become a strength of the members of the APCL.
The Specific Aims for the APCL over the next 5 years are: 1: To provide services in cloning, plasmid construction and over-expression of gene products as requested by the Pi's for use in the Program's various projects. 2: To develop and implement customized protocols uniquely optimized for the purification of the over-expressed proteins to be used in proposed studies. 3: To characterize expressed and purified proteins in terms of purity, elements of structure, and dispersity in solution as preliminary indicators of proper folding. 4: To provide somatic antigen (LPS) as requested for use by the investigators in their individual studies. The expertise and services provided within this Core are fundamental to all aspects of the proposed research and the convenience of the APCL will synergistically enhance the proposed outcomes by providing a consistent quality pool of antigens made by investigators who are experts in the production of such macromolecules.
significant problem encountered in research on human pathogens is the need to generate purified forms of the molecules that are recognized by the innate and/or acquired immune response. The macromolecules required for such research are not available commercially and there is no easy formula for preparing these compounds. Thus, the centralization of antigen preparation into a single Core avoids needless redundancies in the proposed investigations and thus synergistically contributes to the goals of the overall program.
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