Highly Branched Poly(N-isopropylacrylamide) for Use in Protein Purification

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• 作者：Steven Carter ; Stephen Rimmer ; Ramune Rutkaite ; Linda Swanson ; J. P. A. Fairclough ; Alice Sturdy ; and Michelle Webb
• 刊名：Biomacromolecules
• 出版年：2006
• 出版时间：April 2006
• 年：2006
• 卷：7
• 期：4
• 页码：1124 - 1130
• 全文大小：304K
• 年卷期：v.7,no.4(April 2006)
• ISSN：1526-4602

文摘

Poly(N-isopropylacrylamide)s with imidazole endgroups were used to separate a histidine-tagged protein fragmentdirectly from a crude cell lysate. The polymers display a lower critical solution temperature that can be tuned tooccur at a range of subambient temperatures. UV-visible spectra indicated differences in the binding in aqueousmedia of Cu(II) and Ni(II) to the imidazole endgroups. These changes in the UV-visible spectra were reflectedin the solution/aggregation behavior of the polymers as studied by dynamic light scattering. The addition ofCu(II) disaggregated the polymers, and the polymer coil swelled. On the other hand, when Ni(II) was added thepolymers remained aggregated in aqueous media. The polymers were used to purify residues 230-534 of thehistidine-tagged breast cancer susceptibility protein his₆-BRCA1. Cu(II) was found to be better suited to theformation of useful polymer-metal ion-protein complexes that display cloud points, since Ni(II)/polymer mixturesgenerated very little purified protein. The polymers were synthesized using a previously reported variation of thereversible addition-fragmentation chain termination (RAFT) methodology, using the chain transfer agent 3H-imidazole-4-carbodithioic acid 4-vinyl benzyl ester with N-isopropylacrylamide (NIPAM).