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 1.1 mg of EGFP, a value that extrapolates to 50 mg per liter of cell culture. The results suggest that Acanthamoeba is a useful cost-effective system for the production of recombinant proteins. (C) 2009 Elsevier Inc. All rights reserved.