Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote Aspergillus nidulans.
Despite detailed genetic and mutagenic analysis and a recent high-resolution structure of a bacterial member of the nucleobase-ascorbate transporter (NAT) family, understanding of the mechanism of action of eukaryotic NATs is limited. Preliminary studies successfully expressed and purified wild-type UapA to high homogeneity; however, the protein was extremely unstable, degrading almost completely after 48 h at 4°C. In an attempt to increase UapA stability we generated a number of single point mutants (E356D, E356Q, N409A, N409D, Q408E and G411V) previously shown to have reduced or no transport activity, but correct targeting to the membrane. The mutant UapA constructs expressed well as GFP fusions in Saccharomyces cerevisiae and exhibited similar fluorescent size exclusion chromatography (FSEC) profiles to the wild-type protein, following solubilization in 1% DDM, LDAO or OM + 1 mM xanthine. In order to assess the relative stabilities of the mutants, solubilized fractions prepared in 1% DDM + 1 mM xanthine were heated at 45°C for 10 min prior to FSEC. The Q408E and G411V mutants gave markedly better profiles than either wild-type or the other mutants. Further FSEC analysis following solubilization of the mutants in 1% NG ± xanthine confirmed that G411V is more stable than the other mutants, but showed that Q408E is unstable under these conditions. G411V and an N-terminally truncated construct G411VΔ1-11 were submitted to large-scale expression and purification. Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies.
Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, UK.
James
J
Leung
Alexander D
AD
Cameron
George
G
Diallinas
Bernadette
B
Byrne
D001233:Aspergillus nidulans / Q000235:genetics*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D055550:Protein Stability / Q000187:drug effects; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D012995:Solubility; D013696:Temperature; D019820:Xanthine / Q000494:pharmacology
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C083852:UAPA protein, Aspergillus nidulans; D019820:Xanthine