Design of improved membrane protein production experiments: quantitation of the host response.
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 tightly-defined 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.
Department of Cell and Molecular Biology/Microbiology, Göteborg University, Sweden.
Nicklas
N
Bonander
Kristina
K
Hedfalk
Christer
C
Larsson
Petter
P
Mostad
Celia
C
Chang
Lena
L
Gustafsson
Roslyn M
RM
Bill
D019149:Bioreactors; D002450:Cell Communication; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification*; D020411:Oligonucleotide Array Sequence Analysis; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D012331:RNA, Fungal; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins