Overview of the pipeline for structural and functional characterization of macrophage proteins at the university of queensland

Abstract

This chapter describes the methodology adopted in a project aimed at structural and functional characterization of proteins that potentially play an important role in mammalian macrophages. The methodology that underpins this project is applicable to both small research groups and larger structural genomics consortia. Gene products with putative roles in macrophage function are identified using gene expression information obtained via DNA microarray technology. Specific targets for structural and functional characterization are then selected based on a set of criteria aimed at maximizing insight into function. The target proteins are cloned using a modification of Gateway cloning technology, expressed with hexa-histidine tags in E. coli, and purified to homogeneity using a combination of affinity and size exclusion chromatography. Purified proteins are finally subjected to crystallization trials and/or NMR-based screening to identify candidates for structure determination. Where crystallography and NMR approaches are unsuccessful, chemical cross-linking is employed to obtain structural information. This resulting structural information is used to guide cell biology experiments to further investigate the cellular and molecular function of the targets in macrophage biology. Jointly, the data sheds light on the molecular and cellular functions of macrophage proteins.