Isolation and Purification of Actin Homolog MreB from Caulobacter crescentus

Abstract

Caulobacter crescentus is an important model for studying cell cycle regulation, cellular differentiation, and cell polarity. The perceived absence of a cytoskeletal network in bacteria was once considered a defining distinction between prokaryotic and eukaryotic cells. However, orhtologues of all three major classes of eukaryotic cytoskeletal proteins have been identified: FtsZ, a tubulin homolog, MreB and ParM, actin homologs, and CreS (crescentin) which appears to be an intermediate filament protein. MreB is required for the maintenance of the rod cell shape. MreB is organized in an axial spiral that is dynamically rearranged during the cell cycle. Little is known about the mechanisms regulating MreB polymerization. To this end, we aimed to purify MreB for use in in vitro polymerization assays. We used two schemes to purify C. crescentus MreB. The first method employs a cleavable GST‐MreB fusion protein overexpressed in E. coli. The second method takes advantage of buffer conditions that support either polymerization or depolymerization of MreB: MreB transitions between soluble and insoluble states, with purification by ultracentrifugation. Elucidation of MreB polymerization and depolymerization mechanisms has the potential to impact the design of chemotherapeutics affecting cell cycle regulation, cellular differentiation, and cell polarity. Supported by NIH T34GM008411