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New examples of membrane protein expression and purification using the yeast based Pdr1-3 expression strategy

Paper ID Volume ID Publish Year Pages File Format Full-Text
23011 43409 2014 7 PDF Available
Title
New examples of membrane protein expression and purification using the yeast based Pdr1-3 expression strategy
Abstract

•Yeast represents an interesting host for membrane protein expression.•Fast and chromosomal integration of any membrane protein gene is possible.•Mutations in a transcription factor allow constitutive membrane protein expression.•Successful expression of membrane proteins from different families.

Overexpression and purification of membrane proteins has been a bottleneck for their functional and structural study for a long time. Both homologous and heterologous expression of membrane proteins with suitable tags for purification presents unique challenges for cloning and expression. Saccharomyces cerevisiae is a potential host system with significant closeness to higher eukaryotes and provides opportunity for attempts to express membrane proteins. In the past, bakers yeast containing mutations within the transcriptional regulator Pdr1 has been used to overexpress various membrane proteins including for example the ABC transporters Pdr5 and Yor1, respectively. In this study we exploited this system and tried to express and purify 3 membrane proteins in yeast along with Pdr5 and Yor1 viz. Rsb1, Mdl1 and Drs2 by virtue of an N-terminal 14-histidine affinity tag. Out of these five, we could express all membrane proteins although at different levels. Satisfactory yields were obtained for three examples i.e. Pdr5, Yor1 and Drs2. Rsb1 expression was comparatively low and Mdl1 was rather unsatisfactory. Thus, we demonstrate here the application of this yeast based expression system that is suitable for cloning, expression and purification of a wide variety of membrane proteins.

Keywords
ATP binding cassette (ABC) transporters; Multidrug resistance; P-type ATPases; Floppases; Membrane protein overexpressionGPD, gylceraldehyde-3-phosphate dehydrogenase; LTE, lipid translocating enzyme; MDR, multidrug resistance; PDR, pleiotropic drug resis
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New examples of membrane protein expression and purification using the yeast based Pdr1-3 expression strategy
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 158–164
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us