TY - JOUR
T1 - A novel membrane based process to isolate recombinant human chemokine receptor CCR3 produced in Escherichia coli
AU - Liu, Jianguo
AU - Chen, Changzhen
AU - Ge, Baosheng
AU - Lu, Jianren
AU - Cui, Zhanfeng
PY - 2013/1/1
Y1 - 2013/1/1
N2 - 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.
AB - 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.
KW - CC chemokine receptor 3
KW - Membrane protein
KW - Purification
KW - Ultrafiltration
U2 - 10.1016/j.memsci.2012.09.020
DO - 10.1016/j.memsci.2012.09.020
M3 - Article
SN - 1873-3123
VL - 425-426
SP - 98
EP - 104
JO - Journal of Membrane Science
JF - Journal of Membrane Science
ER -