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The isolation of recombinant human chemokine receptor CCR3, which fused to maltose binding protein (MBP), has been conducted using ultrafiltration with 300. kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on MBP-CCR3 stability and transmission were quantified using dot blot analysis and parameter scanning ultrafiltration respectively. These conditions included solution pH, ionic strength, stirring speed, permeate flux, and detergent (Fos choline-14) concentration. Under optimized conditions, the MBP-CCR3 purity obtained in the retentate was about 96% and the recovery of MBP-CCR3 was close to 89% after ultrafiltration. The resulting MBP-CCR3 product was then analyzed by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel eletrophoresis and circular dichroism, to confirm its isoelectric point, molecular weight and molecular secondary structure. To our knowledge, this is the first paper of the isolation of a G protein coupled receptor (GPCR) using ultrafiltration alone. © 2012 Elsevier B.V.

Original publication

DOI

10.1016/j.memsci.2012.09.020

Type

Journal article

Journal

Journal of Membrane Science

Publication Date

01/01/2013

Volume

425-426

Pages

98 - 104