title: Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes produced in a prokaryotic expression system.
abstract: Chemical chaperones are low molecular weight compounds known to stabilize proteins in vitro. Recently it was shown that, in transfected cells, these molecules can also correct the defective folding of some mutant proteins. Hyperphenylalaninemia (HPA) has been proposed to be classified as a "conformational disease," since it has been shown that the majority of the PAH mutations affect protein folding, thereby causing an increasing tendency toward aggregation and proteolytic degradation. Based on these observations, the effect of glycerol as a stabilizer agent of recombinant mutant forms of human phenylalanine hydroxylase enzymes (hPAH) produced in a prokaryotic expression system was investigated. The wild-type and two mutant forms of the hPAH protein (R270K and V388M) were expressed in the presence of glycerol in the culture medium. The yield, specific enzymatic activities, and kinetic properties of the recombinant proteins were determined and compared with the data obtained under normal growth conditions. The results obtained demonstrate that glycerol not only improved the yield of the soluble hPAH proteins (2- to 3-fold depending on the mutant enzyme) produced but also increased the specific activity of the purified recombinant enzymes. We speculate that correction of protein folding abnormalities by chemical chaperones may be a possible therapeutic approach to correct conformational diseases.
authors.affiliation: Centro de Patogénese Molecular, Faculdade Farmácia da UL, R. Prof. Gama Pinto, 1649-003 Lisbon, Portugal.
authors.forename: P
authors.initials: P
authors.lastname: Leandro
authors.forename: M C
authors.initials: MC
authors.lastname: Lechner
authors.forename: I
authors.initials: I
authors.lastname: Tavares de Almeida
authors.forename: D
authors.initials: D
authors.lastname: Konecki
mesh_terms: D019943:Amino Acid Substitution; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology*; D006801:Humans; D009154:Mutation; D010651:Phenylalanine Hydroxylase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010651:Phenylalanine Hydroxylase; D005990:Glycerol