Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris
β-hexosaminidases (Hex) are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by α- and/or β-subunits encoded byHEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders ca...
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Pontificia Universidad Javeriana
2016-08-01
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| Series: | Universitas Scientiarum |
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| Online Access: | http://revistas.javeriana.edu.co/index.php/scientarium/article/view/15736 |
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| author | Angela Johana Espejo Mojica Angela Rocío Mosquera Edwin Alexander Rodríguez-López Dennis Johana Díaz Laura Milena Betrán Francy Liliana Hernández Carlos Javier Alméciga Díaz Luis Alejandro Barrera |
| author_facet | Angela Johana Espejo Mojica Angela Rocío Mosquera Edwin Alexander Rodríguez-López Dennis Johana Díaz Laura Milena Betrán Francy Liliana Hernández Carlos Javier Alméciga Díaz Luis Alejandro Barrera |
| author_sort | Angela Johana Espejo Mojica |
| collection | DOAJ |
| description | β-hexosaminidases (Hex) are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by α- and/or β-subunits encoded byHEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders caused by the accumulation of non-degraded glycolipids. Although tissue-derived Hex have been widely characterized, limited information is available for recombinant β-hexosaminidases.
In this study, human lysosomal recombinant Hex (rhHex-A, rhHex-B, and rhHex-S) were produced in the methylotrophic yeast Pichia pastoris GS115. The highest specific enzyme activities were 13,124 for rhHexA; 12,779 for rhHex-B; and 14,606 U .mg-1
for rhHex-S. These results were 25- to 50-fold higher than those obtained from normal human leukocytes. Proteins were purified and characterized at different pH and temperature conditions. All proteins were stable at acidic pH, and at 4 °C and 37 °C. At 45 °C rhHex-S was completely inactivated, while rhHex-A and
rhHex-B showed high stability. This study demonstrates P. pastoris GS115 potential for polymeric lysosomal enzyme production, and describes the characterization of
recombinant β-hexosaminidases produced within the same host. |
| format | Article |
| id | doaj-art-c0b11ebbfff04d678c4c7e530980e457 |
| institution | OA Journals |
| issn | 0122-7483 2027-1352 |
| language | English |
| publishDate | 2016-08-01 |
| publisher | Pontificia Universidad Javeriana |
| record_format | Article |
| series | Universitas Scientiarum |
| spelling | doaj-art-c0b11ebbfff04d678c4c7e530980e4572025-08-20T01:51:20ZengPontificia Universidad JaverianaUniversitas Scientiarum0122-74832027-13522016-08-0121319521710.11144/Javeriana.SC21-3.corhCharacterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastorisAngela Johana Espejo Mojica0Angela Rocío Mosquera1Edwin Alexander Rodríguez-López2Dennis Johana Díaz3Laura Milena Betrán4Francy Liliana Hernández5Carlos Javier Alméciga Díaz6Luis Alejandro Barrera7Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, ColombiaInstitute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, ColombiaChemistry Department, School of Science, Pontificia Universidad Javeriana, Bogotá, ColombiInstitute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, ColombiaInstitute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, ColombiaInstitute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, ColombiaInstitute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, ColombiaInstitute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana. Bogotá, Colombiaβ-hexosaminidases (Hex) are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by α- and/or β-subunits encoded byHEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders caused by the accumulation of non-degraded glycolipids. Although tissue-derived Hex have been widely characterized, limited information is available for recombinant β-hexosaminidases. In this study, human lysosomal recombinant Hex (rhHex-A, rhHex-B, and rhHex-S) were produced in the methylotrophic yeast Pichia pastoris GS115. The highest specific enzyme activities were 13,124 for rhHexA; 12,779 for rhHex-B; and 14,606 U .mg-1 for rhHex-S. These results were 25- to 50-fold higher than those obtained from normal human leukocytes. Proteins were purified and characterized at different pH and temperature conditions. All proteins were stable at acidic pH, and at 4 °C and 37 °C. At 45 °C rhHex-S was completely inactivated, while rhHex-A and rhHex-B showed high stability. This study demonstrates P. pastoris GS115 potential for polymeric lysosomal enzyme production, and describes the characterization of recombinant β-hexosaminidases produced within the same host.http://revistas.javeriana.edu.co/index.php/scientarium/article/view/15736β-N-acetylhexosaminidases; characterization; Pichia pastoris; recombinant hexosaminidases; Sandhoff diseaseβ-N-acetylhexosaminidasescharacterizationPichia pastorisrecombinant hexosaminidasesSandhoff diseaseTay Sachs disease. |
| spellingShingle | Angela Johana Espejo Mojica Angela Rocío Mosquera Edwin Alexander Rodríguez-López Dennis Johana Díaz Laura Milena Betrán Francy Liliana Hernández Carlos Javier Alméciga Díaz Luis Alejandro Barrera Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris Universitas Scientiarum β-N-acetylhexosaminidases; characterization; Pichia pastoris; recombinant hexosaminidases; Sandhoff disease β-N-acetylhexosaminidases characterization Pichia pastoris recombinant hexosaminidases Sandhoff disease Tay Sachs disease. |
| title | Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris |
| title_full | Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris |
| title_fullStr | Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris |
| title_full_unstemmed | Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris |
| title_short | Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris |
| title_sort | characterization of recombinant human lysosomal beta hexosaminidases produced in the methylotrophic yeast pichia pastoris |
| topic | β-N-acetylhexosaminidases; characterization; Pichia pastoris; recombinant hexosaminidases; Sandhoff disease β-N-acetylhexosaminidases characterization Pichia pastoris recombinant hexosaminidases Sandhoff disease Tay Sachs disease. |
| url | http://revistas.javeriana.edu.co/index.php/scientarium/article/view/15736 |
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