Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

Nicklas Bonander, Richard A.J. Darby, Ljuban Grgic, Nagamani Bora, Jikai Wen, Saverio Brogna, David R. Poyner, Michael A. A. O'Neill, Roslyn M. Bill

Research output: Contribution to journalArticle

Abstract

Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.
LanguageEnglish
Article number10
JournalMicrobial Cell Factories
Volume8
Issue number10
DOIs
Publication statusPublished - 29 Jan 2009

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Ribosome Subunits
Recombinant proteins
Gene Expression Profiling
Recombinant Proteins
Yeast
Yeasts
Membrane Proteins
Eukaryotic Small Ribosome Subunits
Genes
Polyribosomes
Explosions
Doxycycline
Proteins
Ribosomes
Cultured Cells
Phenotype
Temperature
Membranes
Engineers
Tuning

Bibliographical note

© 2009 Bonander et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Keywords

  • recombinant protein
  • eukaryotic protein
  • transcriptome analysis
  • genes
  • membrane protein production experiments
  • yeast strains
  • high-yielding protein production host

Cite this

Bonander, Nicklas ; Darby, Richard A.J. ; Grgic, Ljuban ; Bora, Nagamani ; Wen, Jikai ; Brogna, Saverio ; Poyner, David R. ; O'Neill, Michael A. A. ; Bill, Roslyn M. / Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield. In: Microbial Cell Factories. 2009 ; Vol. 8, No. 10.
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Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield. / Bonander, Nicklas; Darby, Richard A.J.; Grgic, Ljuban; Bora, Nagamani; Wen, Jikai; Brogna, Saverio; Poyner, David R.; O'Neill, Michael A. A.; Bill, Roslyn M.

In: Microbial Cell Factories, Vol. 8, No. 10, 10, 29.01.2009.

Research output: Contribution to journalArticle

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T1 - Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

AU - Bonander, Nicklas

AU - Darby, Richard A.J.

AU - Grgic, Ljuban

AU - Bora, Nagamani

AU - Wen, Jikai

AU - Brogna, Saverio

AU - Poyner, David R.

AU - O'Neill, Michael A. A.

AU - Bill, Roslyn M.

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