title: Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris.
abstract: 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 72h. 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.
authors.affiliation: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Pey Yee
authors.initials: PY
authors.lastname: Lee
authors.affiliation: School of Biosciences, Taylor's University (Lakeside Campus), 47500 Subang Jaya, Selangor, Malaysia.
authors.forename: Voon Chen
authors.initials: VC
authors.lastname: Yong
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Rozita
authors.initials: R
authors.lastname: Rosli
authors.affiliation: School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia.
authors.forename: Lay Harn
authors.initials: LH
authors.lastname: Gam
authors.affiliation: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Electronic address: cpp_labs@yahoo.com.
authors.forename: Pei Pei
authors.initials: PP
authors.lastname: Chong
mesh_terms: D041022:Candida tropicalis / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D010843:Pichia / Q000235:genetics*
chemical_list: D013186:Farnesyl-Diphosphate Farnesyltransferase
keywords: AOX1; Antigen; BMGY; BMMY; BSA; Candida tropicalis; DTT; EDTA; LiCl; PBS; PMSF; PVDF; Pichia pastoris; Recombinant protein; SS; Squalene synthase; TBST; Tris-buffered saline with 0.05% Tween 20; YPD; alcohol oxidase 1; base pair; bovine serum albumin; bp; buffered glycerol-complex medium; buffered methanol-complex medium; dithiothreitol; ethylenediaminetetraacetic acid; lithium chloride; phenylmethylsulfonyl fluoride; phosphate-buffered saline; polyvinylidene difluoride; squalene synthase; yeast extract-peptone-dextrose