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Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris


Lee, Pey Yee and Yong, Voon Chen and Rosli, Rozita and Gam, Lay Harn and Chong, Pei Pei (2014) Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris. Protein Expression and Purification, 94. pp. 15-21. ISSN 1046-5928; ESSN: 1096-0279


Squalene synthase (SS) is the key precursor and first committed enzyme of the sterol biosynthesis pathway. In a previous work, SS has been identified as one of the immunogenic proteins that could be a potential diagnostic candidate for the pathogenic fungus Candida tropicalis. In this study, SS from C. tropicalis was cloned and expressed as recombinant protein in Pichia pastoris to investigate its reactivity with serum antibodies. ERG9 gene that encodes for SS was amplified by PCR and cloned in-frame into pPICZB expression vector. The recombinant construct was then transformed into P. pastoris GS115 host strain. Expression of the recombinant protein was confirmed by SDS–PAGE and Western blot analysis using anti-His tag probe. Optimal protein production was achieved by cultivating the culture with 1.0% methanol for 72 h. The recombinant protein was purified to approximately 97% pure in a single step immobilized metal affinity chromatography with a yield of 70.3%. Besides, the purified protein exhibited specific reactivity with immune sera on Western blot. This is the first report on heterologous expression of antigenic SS from C. tropicalis in P. pastoris which can be exploited for large-scale production and further research. The results also suggested that the protein might be of great value as antigen candidate for serodiagnosis of Candida infection.

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Additional Metadata

Item Type: Article
Divisions: Faculty of Medicine and Health Science
DOI Number: https://doi.org/10.1016/j.pep.2013.10.012
Publisher: Elsevier
Keywords: Antigen; Candida tropicalis; Pichia pastoris; Recombinant protein; Squalene synthase
Depositing User: Nabilah Mustapa
Date Deposited: 24 May 2015 03:03
Last Modified: 01 Oct 2015 02:24
Altmetrics: http://www.altmetric.com/details.php?domain=psasir.upm.edu.my&doi=10.1016/j.pep.2013.10.012
URI: http://psasir.upm.edu.my/id/eprint/36798
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