Integral Membrane Protein Expression in Saccharomyces cerevisiae

Boswell-Casteel, Rebba C.; Johnson, Jennifer M.; Stroud, Robert M.; Hays, Franklin A.

doi:10.1007/978-1-4939-3637-3_11

Cited by 3 publications

(1 citation statement)

References 13 publications

(18 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After cell disruption, the membrane protein fraction was collected by ultracentrifugation. It is generally recommended to pellet membrane proteins at 100,000–200,000× g for 1- 2 h 43 – 45 , 48 . We investigated the time needed to pellet recombinant Dv CH3H at 200,000× g by analysing samples after 30 min and 60 min.…”

Section: Resultsmentioning

confidence: 99%

First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin

Hausjell

Weissensteiner

Molitor

et al. 2022

Sci Rep

View full text Add to dashboard Cite

Anthochlor pigments (chalcones and aurones) play an important role in yellow flower colourization, the formation of UV-honey guides and show numerous health benefits. The B-ring hydroxylation of chalcones is performed by membrane bound cytochrome P450 enzymes. It was assumed that usual flavonoid 3′-hydroxlases (F3′Hs) are responsible for the 3,4- dihydroxy pattern of chalcones, however, we previously showed that a specialized F3′H, namely chalcone 3-hydroxylase (CH3H), is necessary for the hydroxylation of chalcones. In this study, a sequence encoding membrane bound CH3H from Dahlia variabilis was recombinantly expressed in yeast and a purification procedure was developed. The optimized purification procedure led to an overall recovery of 30% recombinant DvCH3H with a purity of more than 84%. The enzyme was biochemically characterized with regard to its kinetic parameters on various substrates, including racemic naringenin, as well as its enantiomers (2S)-, and (2R)-naringenin, apigenin and kaempferol. We report for the first time the characterization of a purified Cytochrome P450 enzyme from the flavonoid biosynthesis pathway, including the transmembrane helix. Further, we show for the first time that recombinant DvCH3H displays a higher affinity for (2R)-naringenin than for (2S)-naringenin, although (2R)-flavanones are not naturally formed by chalcone isomerase.

show abstract

Section: Resultsmentioning

confidence: 99%

First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin

Hausjell

Weissensteiner

Molitor

et al. 2022

Sci Rep

View full text Add to dashboard Cite

show abstract

Overproduction of Membrane-Associated, and Integrated, Proteins Using Saccharomyces cerevisiae

Haslem

Brown

Zhang

et al. 2022

Methods in Molecular Biology

View full text Add to dashboard Cite

Challenges and Solutions in the Recombinant Expression of Membrane Proteins

Liu,

He,

Tian

et al. 2023

PPL

View full text Add to dashboard Cite

Membrane proteins are important components of the proteome and play key roles in many biological processes, such as signal transduction, material transport, cell recognition, etc. Membrane proteins are involved in several fields, and more and more researchers want to understand them. However, the structural properties of membrane proteins make their recombinant expression yield low. This adversely affects the study of the structure and function of membrane proteins. Therefore, it is crucial to have a comprehensive and up-to-date understanding of membrane protein recombinant expression. Based on the current stage of research on membrane proteins, the article describes the current challenges faced by membrane protein recombinant expression and the solutions that can be applied to lay the foundation for a better study of membrane proteins in the future.

show abstract

Integral Membrane Protein Expression in Saccharomyces cerevisiae

Cited by 3 publications

References 13 publications

First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin

First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin

Overproduction of Membrane-Associated, and Integrated, Proteins Using Saccharomyces cerevisiae

Challenges and Solutions in the Recombinant Expression of Membrane Proteins

Contact Info

Product

Resources

About