Expression, purification and characterization of recombinant human alphagalactosidase A mutants

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress.; 2020

Fabry disease is a recessive X-linked lisosomal storage disorder caused by a deficiency of thealpha-galactosidase A (GLA). This disorder can be treated using enzyme replacement therapywith recombinant GLA. However, there are several limitations in this type of therapy such as thehigh amounts of enzyme required and the cost associated, the possible unwanted immunereactions, and the stability of the enzyme, among others. To overcome some of these limitations,an alternative could be the use of an enzyme with higher specific activity or an enzyme with ahigher half-life. In this work we analyze the enzymatic activity of three point mutations (C90T,N139S and R252T), combinations thereof [C90T plus N139S (C-N), C90T plus R252T (C-R)and N139S plus R252T (N-R)] and two deletion mutants of GLA (Δ7 and Δ10, having 7 or 10amino acids deletions at the C-terminal end, respectively). All recombinant proteins contain ahis-tag fused at the C-terminal of the GLA. The wild type GLA and mutant derivatives wereexpressed using a transient expression system in HEK293 cells and then purified by affinitychromatography from culture supernatant. The analysis of the culture supernatant and purifiedproteins showed that C90T, N139S, C-N and C-R point mutants displayed a lower specificenzyme activity compared with WT enzyme. However, R252T, R-N, Δ7 and Δ10 mutantsdisplayed an increased specific activity compared with WT GLA. More interesting, the last fourmutants showed an increased stability, compared with WT enzyme, after storage in a non-optimal buffer at 4°C, being the Δ7 derivative the most stable. WT enzyme maintained less than1% of its enzyme activity while Δ7 derivative maintained more than 80% of its enzyme activityafter 8 days under this storage condition. These results suggest that some point or deletionmutants of the GLA enzyme could be considered as potentially more efficient for the Fabryenzyme replacement therapy.