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...

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Bibliographic Details
Published in:Indian journal of biochemistry & biophysics 2008-12, Vol.45 (6), p.374-378
Main Authors: Lee, Seonmin, Han, Xuezhe, Choi, Kyu Jin, Ding, Yan, Choi, Taegyu, Tak, Eunyoung, Lee, Jisun, Ha, Joohun, Kim, Sung Soo, Lee, Jinhwa
Format: Article
Language:English
Subjects:
Animals
Cloning, Molecular
Cyclophilin A - genetics
Escherichia coli - enzymology
Formates - chemistry
Glutathione Transferase - genetics
Molecular Chaperones - genetics
Protein Binding
Protein Folding
Rats
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - isolation & purification
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Summary: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.
ISSN:0301-1208
0975-0959