title: Expression plasmids and production of EGFP in stably transfected Acanthamoeba.
abstract: New plasmids containing the TATA-Binding Protein (TBP), TBP Promoter Binding Factor (TPBF) or Glyceraldehyde Phosphate Dehydrogenase (GAPDH) gene promoters from Acanthamoeba castellanii are described. The promoters for Acanthamoeba TPBF and GAPDH genes were used to drive constitutive expression of enhanced green fluorescent protein (EGFP) in stably transfected Acanthamoeba. Based initially on fluorescence microscopy and SDS-PAGE analysis of EGFP, both promoters produce robust expression of EGFP, with the highest level obtained from the GAPDH gene promoter in cells grown in low concentrations of neomycin G418. Purification of EGFP from lysates of 22-ml cultures by conventional chromatography yielded approximately 1.1mg of EGFP, a value that extrapolates to 50mg per liter of cell culture. The results suggest that Acanthamoeba is a useful cost-effective system for the production of recombinant proteins.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA. ebateman@uvm.edu
authors.forename: Erik
authors.initials: E
authors.lastname: Bateman
mesh_terms: D000048:Acanthamoeba / Q000235:genetics* / Q000378:metabolism; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; C097052:TPBF protein, Acanthamoeba castellanii; D015534:Trans-Activators; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)