This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Previously, we described unexpected difficulties we encountered in expression of chemokine genes using our initial strategies for expression from T7 vectors in E. coli. Last year, we outlined a plan for expression of the chemokine genes as fusions to a synthetic domain of staphylococcal protein A, termed 'Z'. We produced and tested vectors that were designed to express both intracellular and secreted Z-chemokine fusions; though both classes of vectors produced recombinant proteins, the constructs with the secretion signals failed to be secreted, so we have chosen to abandon them and to work exclusively with the vectors designed for intracellular expression. Expression from these constructs is in the form of inclusion bodies, and we are now developing conditions for folding, purification, cleavage of the protein A fusion tag, and characterization
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