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

Siyavuya Ishmael Bulani,Ntsane Moleleki,Lucy Moleleki,Jacobus Albertyn

doi:10.1186/2191-0855-2-27

Siyavuya Ishmael Bulani, Ntsane Moleleki + Show 2 more

Open Access

https://doi.org/10.1186/2191-0855-2-27

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Abstract

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.

Highlights

Since the first development of a cell surface display system on bacteriophage by Smith (1985), various yeast cell surface displaying systems have been developed for expression of heterologous proteins (Sergeeva et al 2006)
For displaying mCherry on the cell surface of Y. lipolytica, its encoding sequence containing an enterokinase cleavage site at its C-terminal was fused to the N-terminal of the YlCWP1 encoding sequence
The mcherry and YlCWP1 fusion gene was linked by an enterokinase cleavage site (DDDDK) which is essential for cleavage of mCherry protein from the yeast cell surface after expression

Summary

Introduction

Since the first development of a cell surface display system on bacteriophage by Smith (1985), various yeast cell surface displaying systems have been developed for expression of heterologous proteins (Sergeeva et al 2006). Yeast cell surface display has been used as a method of choice for expression of heterologous proteins. Cell surface displaying systems in yeast, in particular Saccharomyces cerevisiae, have been studied. The zeta based plasmid employs the C-terminal end of the YlCWP1 for cell surface display of proteins. A wide range of heterologous proteins have been successfully displayed on Y. lipolytica cell surface using the zeta-based displaying plasmid (Ni et al 2009; Liu et al 2009, 2010; Yu et al 2010). All identified proteins were used successfully to display active Lip lipase on Y. lipolytica employing a zeta-based plasmid. The displaying plasmid of Yue et al (2007) has been used to carry FLO1 for cell surface display of an active mannanase on Y. lipolytica cells (Yang et al 2009)

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