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Logo of ambexSpringerOpen.comThis journalSubmit a manuscriptRegisterSpringerOpen.comAMB Express
 
AMB Express. 2012; 2: 27.
Published online May 20, 2012. doi:  10.1186/2191-0855-2-27
PMCID: PMC3441212
Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica
Siyavuya Ishmael Bulani,corresponding author1,3 Lucy Moleleki,2 Jacobus Albertyn,3 and Ntsane Moleleki1
1Council for Scientific and Industrial Research, CSIR Bioscences, P.O. Box 395, Pretoria, 0001, South Africa
2Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0001, South Africa
3Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, P.O. Box 339, Bloemfontein, 9300, South Africa
corresponding authorCorresponding author.
Siyavuya Ishmael Bulani: sbulani/at/csir.co.za; Lucy Moleleki: lucy.moleleki/at/up.ac.za; Jacobus Albertyn: Albertynj/at/ufs.ac.za; Ntsane Moleleki: ntsane.moleleki/at/yahoo.co.uk
Received March 20, 2012; Accepted May 1, 2012.
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.
Keywords: mCherry, rDNA vector, YlCWP1, Yarrowia lipolytica, Cell surface display
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