Expression, purification and characterization of recombinant human alphagalactosidase A mutants

AMARANTO, M.; GODINO, A.; CARREÑO, A.; MENDOZA, R.; BARRA, J. L.; CORCHERO, J. L.

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

SAIB

Fabry disease is a recessive X-linked lysosomal storage disorder caused by a deficiency of the alpha-galactosidase A (GLA). This disorder can be treated using enzyme replacement therapy with recombinant GLA. However, there are several limitations in this type of therapy such as the high amounts of enzyme required and the associated cost, the possible unwanted immune reactions, and the stability of the enzyme, among others. To overcome some of these limitations, an alternative could be the use of mutant enzymes with a higher specific activity or with a higher half-life. In this work we produced and analyzed 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 10 amino acids deletions at the C-terminal end, respectively). All these recombinant proteins contain a C-terminal His-tag fusion. The wild type GLA and mutant derivatives were expressed by transient gene expression in a human cell line (HEK 293F cells) and then purified by affinity chromatography from culture supernatants. The analysis of the culture supernatant and purified proteins showed that C90T, N139S, C-N, and C-R mutants displayed a lower specific enzyme activity compared with the WT enzyme. However, R252T, R-N, Δ7, and Δ10 mutants showed increased specific activity compared with WT GLA. More interesting, the last four mutants also showed increased stability, compared with the WT enzyme, after storage in a non-optimal buffer at 4°C, being the Δ7 derivative the most stable. WT enzyme maintained less than 1% of its enzyme activity while Δ7 derivative maintained more than 80% of its enzyme activity after 8 days under this storage condition. These results suggest that some point or deletion mutants of the GLA enzyme could be considered as potentially more efficient drugs for the Fabry enzyme replacement therapy.