Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter.
The methylotrophic yeast H. polymorpha is a popular system for the expression of recombinant proteins using the strong and regulatable methanol oxidase (MOX) promoter. Here we show that the constitutive PMA1 promoter can programme the expression of two heterologous proteins, glucose oxidase and human serum albumin. A constitutive promoter provides a useful additional facility to the H. polymorpha expression system because it allows a simplified fermentation regime, avoids the use of methanol, which is both toxic and an explosive hazard, and allows more flexibility for ectopic gene expression during the course of academic studies. A fragment previously isolated in a promoter screen, using glucose oxidase (GOD) as a reporter gene, was shown to consist of the promoter region and the first 659 bp of the H. polymorpha PMA1 gene, encoding the plasma membrane H(+)-ATPase. When the PMA1 promoter was optimally aligned with the GOD coding region, it produced 185 mg/l glucose oxidase in high cell density fed batch fermentations, whereas in previous experiments using the MOX promoter, a yield of 500 mg/l was recovered. The PMA1 promoter was also used to express recombinant human serum albumin (rHA) in H. polymorpha. In high cell density fermentations the PMA1 promoter produced 460 mg/l rHA, whereas 280 mg/l rHA was obtained using the MOX promoter. Taken together, these experiments show that the HpPMA1 programmes the constitutive expression of recombinant proteins and provides a yield comparable to that from the MOX promoter.
Cox
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Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Mead
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Sudbery
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Eland
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Mannazzu
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Evans
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D000429:Alcohol Oxidoreductases / Q000235:genetics; D015153:Blotting, Western; D004271:DNA, Fungal / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005949:Glucose Oxidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D006180:Proton-Translocating ATPases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D004271:DNA, Fungal; D004274:DNA, Recombinant; C434364:PMA2 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D012709:Serum Albumin; D000429:Alcohol Oxidoreductases; D005949:Glucose Oxidase; C029293:methanol oxidase; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases