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

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...

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Main Authors: Lee, Pey Yee, Yong, Voon Chen, Rosli, Rozita, Gam, Lay Harn, Chong, Pei Pei
Format: Article
Language:English
Published: Elsevier 2014
Online Access:http://psasir.upm.edu.my/id/eprint/36798/1/Cloning.pdf
http://psasir.upm.edu.my/id/eprint/36798/
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spelling my.upm.eprints.367982015-10-01T02:24:14Z http://psasir.upm.edu.my/id/eprint/36798/ Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris Lee, Pey Yee Yong, Voon Chen Rosli, Rozita Gam, Lay Harn Chong, Pei Pei 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. Elsevier 2014-02 Article PeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/36798/1/Cloning.pdf 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 10.1016/j.pep.2013.10.012
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description 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.
format Article
author Lee, Pey Yee
Yong, Voon Chen
Rosli, Rozita
Gam, Lay Harn
Chong, Pei Pei
spellingShingle Lee, Pey Yee
Yong, Voon Chen
Rosli, Rozita
Gam, Lay Harn
Chong, Pei Pei
Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris
author_facet Lee, Pey Yee
Yong, Voon Chen
Rosli, Rozita
Gam, Lay Harn
Chong, Pei Pei
author_sort Lee, Pey Yee
title Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris
title_short Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris
title_full Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris
title_fullStr Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris
title_full_unstemmed Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris
title_sort cloning, expression and purification of squalene synthase from candida tropicalis in pichia pastoris
publisher Elsevier
publishDate 2014
url http://psasir.upm.edu.my/id/eprint/36798/1/Cloning.pdf
http://psasir.upm.edu.my/id/eprint/36798/
_version_ 1643831832187240448
score 13.211869

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