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Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica

In this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Yarrowia lipolytica using the C-terminal end of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wall protein 1 (YlCWP1). mCherry was used as a model protein to asses...

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Autores principales: Bulani, Siyavuya Ishmael, Moleleki, Lucy, Albertyn, Jacobus, Moleleki, Ntsane
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441212/
https://www.ncbi.nlm.nih.gov/pubmed/22608131
http://dx.doi.org/10.1186/2191-0855-2-27
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author Bulani, Siyavuya Ishmael
Moleleki, Lucy
Albertyn, Jacobus
Moleleki, Ntsane
author_facet Bulani, Siyavuya Ishmael
Moleleki, Lucy
Albertyn, Jacobus
Moleleki, Ntsane
author_sort Bulani, Siyavuya Ishmael
collection PubMed
description In this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Yarrowia lipolytica using the C-terminal end of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wall protein 1 (YlCWP1). mCherry was used as a model protein to assess the efficiency of the constructed plasmid. Y. lipolytica transformants harbouring the expression cassettes showed a purple colour phenotype on selective YNB-casamino plates as compared to control cells indicating that mCherry was displayed on the cells. Expression of mCherry on cells of Y. lipolytica was confirmed by both fluorescent microscopy and flow cytometry. Furthermore, SDS-PAGE analysis and matrix-assisted laser desorption/ionization (MALDI)-time-of (TOF)-mass spectrometry (MS) peptide mass fingerprinting (PMF) confirmed that the protein cleaved from the yeast cells using enterokinase was mCherry. Efficient cleavage of mCherry reported in this work offers an alternative purification method for displayed heterologous proteins on Y. lipolytica cells using the plasmid constructed in this study. The developed displaying system offers great potential for industrial production and purification of heterologous proteins at low cost.
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spelling pubmed-34412122012-09-17 Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica Bulani, Siyavuya Ishmael Moleleki, Lucy Albertyn, Jacobus Moleleki, Ntsane AMB Express Original Article In this study, a novel rDNA based plasmid was developed for display of heterologous proteins on the cell surface of Yarrowia lipolytica using the C-terminal end of the glycosylphosphatidylinositol (GPI) anchored Y. lipolytica cell wall protein 1 (YlCWP1). mCherry was used as a model protein to assess the efficiency of the constructed plasmid. Y. lipolytica transformants harbouring the expression cassettes showed a purple colour phenotype on selective YNB-casamino plates as compared to control cells indicating that mCherry was displayed on the cells. Expression of mCherry on cells of Y. lipolytica was confirmed by both fluorescent microscopy and flow cytometry. Furthermore, SDS-PAGE analysis and matrix-assisted laser desorption/ionization (MALDI)-time-of (TOF)-mass spectrometry (MS) peptide mass fingerprinting (PMF) confirmed that the protein cleaved from the yeast cells using enterokinase was mCherry. Efficient cleavage of mCherry reported in this work offers an alternative purification method for displayed heterologous proteins on Y. lipolytica cells using the plasmid constructed in this study. The developed displaying system offers great potential for industrial production and purification of heterologous proteins at low cost. Springer 2012-05-20 /pmc/articles/PMC3441212/ /pubmed/22608131 http://dx.doi.org/10.1186/2191-0855-2-27 Text en Copyright ©2012 Bulani et al.; licensee Springer. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Bulani, Siyavuya Ishmael
Moleleki, Lucy
Albertyn, Jacobus
Moleleki, Ntsane
Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica
title Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica
title_full Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica
title_fullStr Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica
title_full_unstemmed Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica
title_short Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica
title_sort development of a novel rdna based plasmid for enhanced cell surface display on yarrowia lipolytica
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441212/
https://www.ncbi.nlm.nih.gov/pubmed/22608131
http://dx.doi.org/10.1186/2191-0855-2-27
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