A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase.
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.
Sabogal
Alex
A
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Rio
Donald C
DC
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
D029721:Drosophila Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D019895:Transposases