TY - BOOK

T1 - Recombinant production and purification of ion channels for biomimetic membranes and cryo-EM studies

AU - Preisler, Sarah Spruce

PY - 2019

Y1 - 2019

N2 - Ion channels are responsible for the selective ion permeation across biological membranes, and their openings and closings are regulated by gating mechanisms. Mutating amino acids responsible for gating and by removing excess regulatory domains of selected ion channels, opens the possibility to utilize these ion channels for creating biomimetic membranes with high ion flux and selectivity. These ion selective membranes may then be used to create sustainable energy by reverse electro dialysis (RED), by using a salinity gradient across the membranes to capturing the free energy produced by mixing seawater and river water, and pose huge potential for overcoming the growing pressure on global energy resources. Membrane proteins are inherently difficult to express and purify in large quantities. With the purpose of using ion channels for this biotechnological application, this thesis reports on establishing; a reliable cloning strategy to easily introduce mutations to the ion channels, find a high yield expression platform for producing these membrane proteins, and establish a robust and scalable purification protocol for obtaining large quantities of stable and pure membrane proteins. Using Saccharomyces cerevisiae as the expression platform it was possible to produce and purify the ion channels to homogeneity and we were able show functionality of ion channels purified in detergent. This platform further enabled the structure determination using cryo-EM of the human chloride channel ClC-1.

AB - Ion channels are responsible for the selective ion permeation across biological membranes, and their openings and closings are regulated by gating mechanisms. Mutating amino acids responsible for gating and by removing excess regulatory domains of selected ion channels, opens the possibility to utilize these ion channels for creating biomimetic membranes with high ion flux and selectivity. These ion selective membranes may then be used to create sustainable energy by reverse electro dialysis (RED), by using a salinity gradient across the membranes to capturing the free energy produced by mixing seawater and river water, and pose huge potential for overcoming the growing pressure on global energy resources. Membrane proteins are inherently difficult to express and purify in large quantities. With the purpose of using ion channels for this biotechnological application, this thesis reports on establishing; a reliable cloning strategy to easily introduce mutations to the ion channels, find a high yield expression platform for producing these membrane proteins, and establish a robust and scalable purification protocol for obtaining large quantities of stable and pure membrane proteins. Using Saccharomyces cerevisiae as the expression platform it was possible to produce and purify the ion channels to homogeneity and we were able show functionality of ion channels purified in detergent. This platform further enabled the structure determination using cryo-EM of the human chloride channel ClC-1.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/1pioq0f/alma99123542614405763

M3 - Ph.D. thesis

BT - Recombinant production and purification of ion channels for biomimetic membranes and cryo-EM studies

PB - Department of Biology, Faculty of Science, University of Copenhagen

ER -