Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

BMC Biotechnology

Urban Johanson¹  Per Kjellbom¹  Erik Alexandersson²  Jonas ÅH Danielson¹  Maria Agemark¹  Kristina Nordén¹ 

[1]Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, S-221 00 Lund, Sweden

[2]Institute for Wine Biotechnology, Faculty of AgriSciences Stellenbosch University, Private Bag X1, 7602 Matieland, South Africa

关键词: qPCR;    Major Intrinsic Proteins;    aquaporins;    Pichia pastoris;   

Others  :  1146449
DOI  :  10.1186/1472-6750-11-47

 received in 2010-07-02, accepted in 2011-05-10,  发布年份 2011

【 摘 要 】

Background

When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast Pichia pastoris.

Results

By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.

Conclusions

We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant P. pastoris clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of P. pastoris clones with high expression levels of aquaporins and other classes of membrane proteins.

【 授权许可】

   
2011 Nordén et al; licensee BioMed Central Ltd.

【 图 表 】

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