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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| Format: | Article |
| Language: | English |
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Elsevier
2014
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| Online Access: | http://psasir.upm.edu.my/id/eprint/36798/ http://psasir.upm.edu.my/id/eprint/36798/1/Cloning.pdf |
| _version_ | 1848848432479862784 |
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| author | Lee, Pey Yee Yong, Voon Chen Rosli, Rozita Gam, Lay Harn Chong, Pei Pei |
| author_facet | Lee, Pey Yee Yong, Voon Chen Rosli, Rozita Gam, Lay Harn Chong, Pei Pei |
| author_sort | Lee, Pey Yee |
| building | UPM Institutional Repository |
| collection | Online Access |
| 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. |
| first_indexed | 2025-11-15T09:34:24Z |
| format | Article |
| id | upm-36798 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T09:34:24Z |
| publishDate | 2014 |
| publisher | Elsevier |
| recordtype | eprints |
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| spelling | upm-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 |
| 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 |
| title | 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_short | 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 |
| url | http://psasir.upm.edu.my/id/eprint/36798/ http://psasir.upm.edu.my/id/eprint/36798/ http://psasir.upm.edu.my/id/eprint/36798/1/Cloning.pdf |