Journal
MACROMOLECULAR RAPID COMMUNICATIONS
Volume 31, Issue 19, Pages 1691-1695Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/marc.201000333
Keywords
glutathione; polyNIPAAm; protein purification; RAFT polymerization; thermoprecipitation
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Funding
- National Science Foundation [CHE-0809832]
- California NanoSystems Institute
- Alfred P Sloan Foundation
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Herein, we report an effective and rapid method to purify glutathione S-transferase (GST) using glutathione (GSH)-modified poly(N-isopropylacrylamide) (pNIPAAm) and mild, thermal conditions. A chain transfer agent modified with pyridyl disulfide was employed in the reversible addition-fragmentation chain transfer (RAFT) polymerization of NIPAAm. The resulting polymer had a narrow molecular weight distribution (polydispersity index = 1.21). Conjugation of GSH to the pyridyl disulfide-pNIPAAm reached 95% within 30 min as determined by UV-Vis monitoring of the release of pyridine-2-thione. GST was successfully thermoprecipitated upon heating the GSH-pNIPAAm above the lower critical solution temperature (LCST). The pull down assay was repeated with bovine serum albumin (BSA) and T4 lysozyme (T4L), which demonstrated the specificity of the polymer for GST. Due to its simplicity and high efficiency, this method holds great potential for large-scale purification of GST-tagged proteins.
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