title: A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase.
abstract: Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site-specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP-binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP-binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP-binding residues in the central portion of the transposase coding region. This clone, amino acids 275-409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP-binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N-terminal THAP DNA-binding domain attached to an extended leucine zipper coiled-coil dimerization domain in the P element transposase, precisely delineating the DNA-binding and dimerization activities on the primary sequence. This study highlights the use of a GFP-based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Sabogal
authors.forename: Donald C
authors.initials: DC
authors.lastname: Rio
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D018983:DNA Footprinting; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D019895:Transposases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D019895:Transposases