Design of improved membrane protein production experiments: quantitation of the host response

Nicklas Bonander, Kristina Hedfalk, Christer Larsson, Petter Mostad, Celia Chang, Lena Gustafsson, Roslyn M. Bill*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightlydefined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology. Copyright © 2005 The Protein Society.

Original languageEnglish
Pages (from-to)1729-1740
Number of pages12
JournalProtein Science
Volume14
Issue number7
DOIs
Publication statusPublished - Jul 2005

Fingerprint

Membrane Proteins
Experiments
Yeast
Growth
Yeasts
Physiology
Bioreactors
Genomics
Recombinant Proteins
Structural analysis
Genes
Research Personnel
Gene Expression
Glucose
Messenger RNA

Keywords

  • expression systems
  • membrane proteins
  • miniarray
  • protein production
  • yeast

Cite this

Bonander, N., Hedfalk, K., Larsson, C., Mostad, P., Chang, C., Gustafsson, L., & Bill, R. M. (2005). Design of improved membrane protein production experiments: quantitation of the host response. Protein Science , 14(7), 1729-1740. https://doi.org/10.1110/ps.051435705
Bonander, Nicklas ; Hedfalk, Kristina ; Larsson, Christer ; Mostad, Petter ; Chang, Celia ; Gustafsson, Lena ; Bill, Roslyn M. / Design of improved membrane protein production experiments : quantitation of the host response. In: Protein Science . 2005 ; Vol. 14, No. 7. pp. 1729-1740.
@article{e6a206d8e5344d65ab7a2df56d44e993,
title = "Design of improved membrane protein production experiments: quantitation of the host response",
abstract = "Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightlydefined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology. Copyright {\circledC} 2005 The Protein Society.",
keywords = "expression systems, membrane proteins, miniarray, protein production, yeast",
author = "Nicklas Bonander and Kristina Hedfalk and Christer Larsson and Petter Mostad and Celia Chang and Lena Gustafsson and Bill, {Roslyn M.}",
year = "2005",
month = "7",
doi = "10.1110/ps.051435705",
language = "English",
volume = "14",
pages = "1729--1740",
journal = "Protein Science",
issn = "0961-8368",
publisher = "Cold Spring Harbor Laboratory Press",
number = "7",

}

Bonander, N, Hedfalk, K, Larsson, C, Mostad, P, Chang, C, Gustafsson, L & Bill, RM 2005, 'Design of improved membrane protein production experiments: quantitation of the host response', Protein Science , vol. 14, no. 7, pp. 1729-1740. https://doi.org/10.1110/ps.051435705

Design of improved membrane protein production experiments : quantitation of the host response. / Bonander, Nicklas; Hedfalk, Kristina; Larsson, Christer; Mostad, Petter; Chang, Celia; Gustafsson, Lena; Bill, Roslyn M.

In: Protein Science , Vol. 14, No. 7, 07.2005, p. 1729-1740.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Design of improved membrane protein production experiments

T2 - quantitation of the host response

AU - Bonander, Nicklas

AU - Hedfalk, Kristina

AU - Larsson, Christer

AU - Mostad, Petter

AU - Chang, Celia

AU - Gustafsson, Lena

AU - Bill, Roslyn M.

PY - 2005/7

Y1 - 2005/7

N2 - Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightlydefined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology. Copyright © 2005 The Protein Society.

AB - Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightlydefined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology. Copyright © 2005 The Protein Society.

KW - expression systems

KW - membrane proteins

KW - miniarray

KW - protein production

KW - yeast

UR - http://www.scopus.com/inward/record.url?scp=22244478668&partnerID=8YFLogxK

UR - http://onlinelibrary.wiley.com/doi/10.1110/ps.051435705/abstract

U2 - 10.1110/ps.051435705

DO - 10.1110/ps.051435705

M3 - Article

C2 - 15987902

VL - 14

SP - 1729

EP - 1740

JO - Protein Science

JF - Protein Science

SN - 0961-8368

IS - 7

ER -

Bonander N, Hedfalk K, Larsson C, Mostad P, Chang C, Gustafsson L et al. Design of improved membrane protein production experiments: quantitation of the host response. Protein Science . 2005 Jul;14(7):1729-1740. https://doi.org/10.1110/ps.051435705