Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes produced in a prokaryotic expression system

Chemical chaperones are low molecular weight compounds known to stabilize p roteins in vitro. Recently it was shown that, in transfected cells, these m olecules can also correct the defective folding of some mutant proteins. Hy perphenylalaninemia (HPA) has been proposed to be classified as a "conforma tional disease," since it has been shown that the majority of the PAH mutat ions affect protein folding, thereby causing an increasing tendency toward aggregation and proteolytic degradation. Based on these observations, the e ffect of glycerol as a stabilizer agent of recombinant mutant forms of huma n phenylalanine hydroxylase enzymes (hPAH) produced in a prokaryotic expres sion system was investigated. The wild-type and two mutant forms of the hPA H protein (R270K and V388M) were expressed in the presence of glycerol in t he culture medium. The yield, specific enzymatic activities, and kinetic pr operties of the recombinant proteins were determined and compared with the data obtained under normal growth conditions. The results obtained demonstr ate 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 t he 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. (C) 200 1 Academic Press.