A new method for purification of functional recombinant GST-cyclophilin A protein from E. coli.
The expression of glutathione-S-transferase (GST) fusion protein is extensively utilized in the study of protein-protein interactions. In the commonly used purification method, the overexpressed GST fusion protein is bound to the glutathione (GSH)-coupled resins via affinity chromatography, and then eluted by an excessive quantity of reduced GSH. However, this technique has certain limitations, such as low product purity, retention of GSH in the sample, as well as relatively high cost. To overcome these limitations, in this study, elution buffer containing 2% formic acid was utilized rather than GSH to elute the GST-fusion protein, and thereafter the acidic samples were neutralized using collecting buffer. By using this method, highly purified GST-cyclophilin A (CypA) fusion protein was obtained, without affecting the structural and functional characteristics such as PPIase and chaperone activities. Moreover, the procedure is also cost-effective, due to the low cost of formic acid as compared with GSH.
Lee
Seonmin
S
Department of Biochemistry and Molecular Biology, Medical Science and Engineering Research Center for Bioreaction to Reactive Oxygen Species and Biomedical Science Institute (BK-21), School of Medicine, Kyung Hee University, Seoul 130-701, Korea.
Han
Xuezhe
X
Choi
Kyu Jin
KJ
Ding
Yan
Y
Choi
Taegyu
T
Tak
Eunyoung
E
Lee
Jisun
J
Ha
Joohun
J
Kim
Sung Soo
SS
Lee
Jinhwa
J
D000818:Animals; D003001:Cloning, Molecular; D021984:Cyclophilin A / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology*; D005561:Formates / Q000737:chemistry; D005982:Glutathione Transferase / Q000235:genetics*; D018832:Molecular Chaperones / Q000235:genetics*; D011485:Protein Binding; D017510:Protein Folding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*
D005561:Formates; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C030544:formic acid; D005982:Glutathione Transferase; D021984:Cyclophilin A