title: Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen.
abstract: SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity.
authors.affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, York, UK.
authors.forename: Andrea E
authors.initials: AE
authors.lastname: Rawlings
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Levdikov
authors.forename: Elena
authors.initials: E
authors.lastname: Blagova
authors.forename: Vicki L
authors.initials: VL
authors.lastname: Colledge
authors.forename: Philippe J
authors.initials: PJ
authors.lastname: Mas
authors.forename: James
authors.initials: J
authors.lastname: Tunaley
authors.forename: Ludmila
authors.initials: L
authors.lastname: Vavrova
authors.forename: Keith S
authors.initials: KS
authors.lastname: Wilson
authors.forename: Imrich
authors.initials: I
authors.lastname: Barak
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Hart
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Wilkinson
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015870:Gene Expression; D016296:Mutagenesis; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C071525:FtsZ protein, Bacteria; C030069:spore-specific proteins, Bacillus
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