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Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter

Cox, Helen and Mead, David and Sudbery, Peter E. and Eland, R. Mark and Mannazzu, Ilaria Maria and Evans, Leslie (2000) Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter. Yeast, Vol. 16 (13), p. 1191-1203. eISSN 1097-0061. Article.

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DOI: 10.1002/1097-0061(20000930)16:13<1191::AID-YEA589>3.0.CO;2-2

Abstract

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.

Item Type:Article
ID Code:3823
Status:Published
Refereed:Yes
Uncontrolled Keywords:Hansenula polymorpha, Saccharomyces cerevisiae, constitutive promoter, protein expression, plasma membrane H+ ATPase, PMA1, glucose oxidase, human serum albumin, rHA
Subjects:Area 07 - Scienze agrarie e veterinarie > AGR/16 Microbiologia agraria
Divisions:001 Università di Sassari > 01 Dipartimenti > Scienze ambientali agrarie e biotecnologie agro-alimentari
Publisher:Wiley
eISSN:1097-0061
Deposited On:04 May 2010 11:55

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