Methods for Expression and Purification of Biologically Active Recombinant Progranulin.

Purifying progranulin may be useful in a variety of situations, for example, after it has been mutated or otherwise modified or when working with a species for which commercially produced progranulin is unavailable. A method to express and purify human progranulin is presented. Progranulin is transiently expressed in mammalian cells and isolated from their conditioned medium before purification by reversed-phase high-performance liquid chromatography (RP-HPLC). Human progranulin is used as an example, but the protocol can be applied to any other progranulin protein. Modifications of the expression-purification strategy for metabolic labeling of progranulin and analytical systems based on heparin-affinity chromatography are presented.

Methods to Investigate the Molecular Basis of Progranulin Actions on Brain and Behavior In Vivo Using Knockout Mice.

Currently one of the few molecules that equally excites a neuroscientist, a cancer biologist, an immunologist, and a developmental biologist is progranulin (GRN/Grn)-a pluripotent growth factor that plays key roles in cell survival, proliferation, development, tissue regeneration, inflammation, wound healing, and angiogenesis. However, the molecular pathways associated with GRN signaling involved in these varied physiological processes are not understood. Gene inactivation has been considered as one of the best methods to delineate the biological role of a protein, and gene targeting is a direct means to disrupt a gene's open reading frame and block its expression, for instance, in a mouse. Such a gene knockout animal model also served as an in vivo disease model where loss of gene or its function is thought to be the primary disease mechanism, as is the case with progranulin loss of function in frontotemporal lobar degeneration (FTLD). It is estimated that up to half of the cases of familial, dominant FTLD might be due to GRN haploinsufficiency. To understand the molecular pathways associated with GRN loss, constitutive and conditional progranulin knockout (Grn-/-) mice have also been constructed in several laboratories, including ours. These mice show several disease-characteristic features and suggest that continued studies on the Grn-/- mice would be instructive in the understanding of complex GRN biology in health and disease.