Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin

↓ Direkt till sidans innehåll
↓ Direkt till sidans sekundära innehåll (sidomenyn)

Sökning: id:"swepub:oai:research.chalmers.se:75990fb0-6812-4ee4-9e97-816f18eb17b1" > Constitutively-stre...

1 av 1
Föregående post
Nästa post
Till träfflistan

Cartwright, S. P.Aston University (författare)

Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin

Artikel/kapitelEngelska2017

Förlag, utgivningsår, omfång ...

2017-03-09
Springer Science and Business Media LLC,2017
electronicrdacarrier

Nummerbeteckningar

LIBRIS-ID:oai:research.chalmers.se:75990fb0-6812-4ee4-9e97-816f18eb17b1
https://doi.org/10.1186/s12934-017-0656-2DOI
https://research.chalmers.se/publication/249101URI

Kompletterande språkuppgifter

Språk:engelska
Sammanfattning på:engelska

Ingår i deldatabas

SwePubSwePub

Klassifikation

Ämneskategori:art swepub-publicationtype
Ämneskategori:ref swepub-contenttype

Anmärkningar

Background: We previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3 Delta, srb5 Delta, gcn5 Delta. and yTHCBMS1 (supplemented with 0.5 mu g/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5' untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10-80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A(2a)R) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described. Results: Polysome profiling experiments were used to analyze the translational state of spt3 Delta, srb5 Delta, gcn5 Delta. and yTHCBMS1 (supplemented with 0.5 mu g/mL doxycycline); all but gcn5 Delta. were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31a Delta., rpl22a Delta., ssf1 Delta. and nop1 Delta.) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3 Delta, srb5 Delta, gcn5 Delta. and yTHCBMS1 (supplemented with 0.5 mu g/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2 alpha. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5' UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3. or wild-type cells, suggesting that high-yielding strains are able to bypass 5' uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories. Conclusions: Regulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production.

Ämnesord och genrebeteckningar

NATURVETENSKAP Biologi hsv//swe
NATURAL SCIENCES Biological Sciences hsv//eng
Aquaglyceroporin
Recombinant protein
Fps1 regulation
Saccharomyces cerevisiae
Leaky scanning
Upstream open reading frame
Translation initiation

Biuppslag (personer, institutioner, konferenser, titlar ...)

Darby, R. A. J.Aston University (författare)
Sarkar, D.Aston University (författare)
Bonander, Nicklas,1968Chalmers tekniska högskola,Chalmers University of Technology(Swepub:cth)bonander (författare)
Gross, S. R.Aston University (författare)
Ashe, M. P.University of Manchester (författare)
Bill, RAston University (författare)
Aston UniversityChalmers tekniska högskola (creator_code:org_t)

Sammanhörande titlar

Ingår i:Microbial Cell Factories: Springer Science and Business Media LLC16:11475-2859

Internetlänk

Hitta via bibliotek

Microbial Cell Factories (Sök värdpublikationen i LIBRIS)

Till lärosätets databas

1 av 1
Föregående post
Nästa post
Till träfflistan

Hitta mer i SwePub

Av författaren/redakt...: Cartwright, S. P ...; Darby, R. A. J.; Sarkar, D.; Bonander, Nickla ...; Gross, S. R.; Ashe, M. P.; visa fler...; Bill, R; visa färre...

Om ämnet

NATURVETENSKAP: NATURVETENSKAP; och Biologi

Artiklar i publikationen: Microbial Cell F ...

Av lärosätet: Chalmers tekniska högskola

Sök utanför SwePub

Sök vidare i:: Google; Google Book Search; Google Scholar

Kungliga biblioteket hanterar dina personuppgifter i enlighet med EU:s dataskyddsförordning (2018), GDPR. Läs mer om hur det funkar här.
Så här hanterar KB dina uppgifter vid användning av denna tjänst.

LIBRIS.kb.se