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

Nordén, Kristina LU ; Agemark, Maria LU ; Danielson, Jonas LU ; Alexandersson, Erik LU ; Kjellbom, Per LU and Johanson, Urban LU (2011) In BMC Biotechnology 11.
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Pichia pastoris aquaporins, Major Intrinsic Proteins, qPCR
in
BMC Biotechnology
volume
11
publisher
BioMed Central
external identifiers
  • wos:000291722700001
  • scopus:79955746703
ISSN
1472-6750
DOI
10.1186/1472-6750-11-47
language
English
LU publication?
yes
id
f82b8caf-a7d8-4d27-8bb5-37cccab9d0a7 (old id 2056806)
date added to LUP
2011-07-26 10:30:29
date last changed
2017-11-12 03:48:55
@article{f82b8caf-a7d8-4d27-8bb5-37cccab9d0a7,
  abstract     = {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.},
  author       = {Nordén, Kristina and Agemark, Maria and Danielson, Jonas and Alexandersson, Erik and Kjellbom, Per and Johanson, Urban},
  issn         = {1472-6750},
  keyword      = {Pichia pastoris aquaporins,Major Intrinsic Proteins,qPCR},
  language     = {eng},
  publisher    = {BioMed Central},
  series       = {BMC Biotechnology},
  title        = {Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris},
  url          = {http://dx.doi.org/10.1186/1472-6750-11-47},
  volume       = {11},
  year         = {2011},
}