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| Functional Analysis of Three Promoters from Yeast in Pichia pastoris |
| JIANG Hui-hui1,2, LI Feng-gong2, LU Yi2, RAO Zhi-ming1,2 |
1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China;
2. Lab of Applied Microbiology and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, China |
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Abstract The promoter is an important cis-element for regulation of gene expression,the strength of the promoter function is very important for the expression of target genes. In order to find a better promoter for transcriptional initiation, the experiment compared different promoters in the expression of foreign proteins in Pichia pastoris, using the green fluorescent protein (GFP) as a reporter gene.The promoter of glycerol-3-phosphate dehydrogenase gene(Scgpd) which is the key enzyme in Glycerol synthesis from Saccharomyces cerevisiae was cloned by PCR, and introduced it into pPIC9K and pGAPZB to construct pPIC9K-gfp and pPIC9K-PG,pPGAPZB-gfp and pGPDZB-gfp simultaneously. The recombinant plasmids were transformed into Pichia pastoris GS115 and Pichia pastoris X33 by electroporation. In the medium containing glucose or NaCl with different concentrations for culturing the recombinant strains, GFP was detected by fluorescent microscopy. By the expression of GFP,the fuction of pScgpd and methanol oxidase promoter pAOX1, 3-GPDH promoter pGAP was compaired. Fluorescence microscopy showed pScgpd induced by the performance of hypertonic conditions. The gene gfp was functionally expressed under the control of the promoters in recombinant Pichia pastoris. Furthermore, the expression of the gene gfp at different level was conducted by the different concentrations of glucose or NaCl within a certain range for the recombinant strains. However, compared with the promoter pGAP and pAOX1 corresponding to the host P.pastoris GS115 and P.pastoris X33, the expression level under the control of the promoter pScgpd is still weak.
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Received: 05 January 2011
Published: 27 May 2011
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