A novel membrane based process to isolate recombinant human chemokine receptor CCR3 produced in Escherichia coli

The isolation of recombinant human chemokine receptor CCR3, which fused to maltose binding protein (MBP), has been conducted using ultrafiltration with 300kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on MBP-CCR3 stability and transmissi...

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Bibliographic Details
Published in:Journal of membrane science 2013-01, Vol.425-426 (1), p.98-104
Main Authors: Liu, Jianguo, Chen, Changzhen, Ge, Baosheng, Lu, Jianren, Cui, Zhanfeng
Format: Article
Language:English
Subjects:
artificial membranes
CC chemokine receptor 3
CCR3 receptor
Escherichia coli
gels
humans
ionic strength
isoelectric focusing
isoelectric point
maltose
Membrane protein
mixing
molecular weight
Purification
sodium dodecyl sulfate
Ultrafiltration
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Summary:The isolation of recombinant human chemokine receptor CCR3, which fused to maltose binding protein (MBP), has been conducted using ultrafiltration with 300kDa 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. ► Isolation of human chemokine receptor CCR3 using ultrafiltration was performed. ► Effects of ultrafiltration operating conditions on CCR3 stability were quantified. ► Effects of operating parameters on protein transmission were studied.
ISSN:0376-7388
1873-3123
DOI:10.1016/j.memsci.2012.09.020