![circular dichroism buffer cut off wavelength circular dichroism buffer cut off wavelength](https://els-jbs-prod-cdn.jbs.elsevierhealth.com/cms/attachment/948578f3-f366-4689-ac6d-fd47bb0a0756/gr1_lrg.jpg)
The Circular Dichroism facility was supported by the Wellcome Trust (ref 094232). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was funded by the Wellcome Trust (ref.
![circular dichroism buffer cut off wavelength circular dichroism buffer cut off wavelength](https://www.mdpi.com/ijms/ijms-22-01199/article_deploy/html/images/ijms-22-01199-g001.png)
Received: JAccepted: OctoPublished: November 25, 2015Ĭopyright: © 2015 Ma et al. PLoS ONE 10(11):Įditor: Kornelius Zeth, University of Roskilde, DENMARK (2015) A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.Ĭitation: Ma C, Hao Z, Huysmans G, Lesiuk A, Bullough P, Wang Y, et al.
#Circular dichroism buffer cut off wavelength zip
To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment.
![circular dichroism buffer cut off wavelength circular dichroism buffer cut off wavelength](https://jascoinc.com/wp-content/uploads/2020/01/thermal-melting-curve-462x480.png)
For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs.