Premium
Thermoprecipitation of Glutathione S ‐Transferase by Glutathione–Poly( N ‐isopropylacrylamide) Prepared by RAFT Polymerization
Author(s) -
Chang ChienWen,
Nguyen Thi H.,
Maynard Heather D.
Publication year - 2010
Publication title -
macromolecular rapid communications
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.348
H-Index - 154
eISSN - 1521-3927
pISSN - 1022-1336
DOI - 10.1002/marc.201000333
Subject(s) - raft , chain transfer , lower critical solution temperature , dispersity , glutathione , chemistry , polymerization , poly(n isopropylacrylamide) , polymer chemistry , lysozyme , reversible addition−fragmentation chain transfer polymerization , bovine serum albumin , polymer , radical polymerization , chromatography , copolymer , organic chemistry , biochemistry , enzyme
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.