Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost

Peti, Wolfgang; Page, Rebecca

doi:10.1016/j.pep.2006.06.024

Cited by 194 publications

(139 citation statements)

References 43 publications

Supporting

Mentioning

137

Contrasting

Unclassified

Order By: Relevance

“…Details are provided in Appendix Supplementary Methods. MBP‐tagged (Peti & Page, 2007) FUS LC and FUS full‐length constructs were purified by immobilized metal ion affinity chromatography followed by gel filtration. MBP‐LCs were cleaved by TEV protease and further purified to leave FUS LC with no remaining tags.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

et al. 2017

View full text Add to dashboard Cite

Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation‐prone, yeast prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.

show abstract

Section: Methodsmentioning

confidence: 99%

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Proper and efficient protein folding might require specific cofactors in the growth media such as metal ions. Addition of these essential factors to the culture media could considerably increase the yield as well as the folding rate of the soluble proteins [4,17] .…”

Section: Modification Of Media Compositionmentioning

confidence: 99%

“…The increase of expression and activity of lower temperatures growth is associated with increased expression of chaperones in E. coli. Therefore, growth at a temperature range of (15)(16)(17)(18)(19)(20)(21)(22)(23) 曟, could also lead to a significant reduction of expressed protein degradation [4,18,19] .…”

Section: Expression At Lower Temperaturesmentioning

confidence: 99%

Strategies for production of active eukaryotic proteins in bacterial expression system

Khow

Suntrarachun

2012

Asian Pacific Journal of Tropical Biomedicine

112

View full text Add to dashboard Cite

“…A strong influence of the fusion partner on protein expression and solubility has been described repeatedly [36][37][38]. In order to facilitate testing for solubility of the produced protein, an additional centrifugation step using filter plates or an ultracentrifugation step using 96 well plates may also be included to remove insoluble material [38][39][40].…”

Section: Discussionmentioning

confidence: 99%

Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen

Mühle

Löchelt

Denner

2012

Protein Expression and Purification

View full text Add to dashboard Cite

The production of recombinant transmembrane proteins is due to their biochemical properties often troublesome and time consuming. Here the prokaryotic expression and purification of the transmembrane envelope proteins of the feline and primate foamy virus using a screening assay for optimisation of expression in 96 deep well plates is described. Testing simultaneously various bacterial strains, media, temperatures, inducer concentrations and different transformants, conditions for an about twentyfold increased production were quickly determined. These small scale test conditions could be easily scaled up, allowing purification of milligram amounts of recombinant protein. Proteins with a purity of about 95% were produced using a new purification protocols, they were characterised by gel filtration and circular dichroism and successfully applied in immunological assays screening for foamy virus infection and in immunisation studies. Compared to the previously described protocol (Muehle et al., Virology, 412:333-340, 2011), proteins with similar characteristics but about thirtyfold increased yields were obtained. The screening and production method presented here can also be applied for the production of transmembrane envelope proteins of other retroviruses, including HIV-1.

show abstract

Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost

Cited by 194 publications

References 43 publications

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

Strategies for production of active eukaryotic proteins in bacterial expression system

Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen

Contact Info

Product

Resources

About