Abstract
Affinity precipitation of proteins uses polymers capable of reversible soluble-insoluble transitions in response to small
environmental changes (temperature, pH or solvent composition). Here we describe protocols for (i) the synthesis of
responsive polymers with specific affinity to target proteins and (ii) the purification of proteins using these polymers.
The purification is based on precipitation of the affinity complex between the protein and the polymer, which is induced
by environmental changes. This separation strategy is simpler and more cost effective than conventional affinity column
chromatography. Specifically, we describe the synthesis of thermoresponsive 1-vinylimidazole:N-isopropylacrylamide
copolymers. The whole procedure takes 2–3 h when applied to purification of recombinant His-tag proteins or proteins with
natural metal binding groups by means of metal chelate affinity precipitation. Optimization of the polymer composition and
the type of chelating ions allows for target protein yields of 80% and higher.
environmental changes (temperature, pH or solvent composition). Here we describe protocols for (i) the synthesis of
responsive polymers with specific affinity to target proteins and (ii) the purification of proteins using these polymers.
The purification is based on precipitation of the affinity complex between the protein and the polymer, which is induced
by environmental changes. This separation strategy is simpler and more cost effective than conventional affinity column
chromatography. Specifically, we describe the synthesis of thermoresponsive 1-vinylimidazole:N-isopropylacrylamide
copolymers. The whole procedure takes 2–3 h when applied to purification of recombinant His-tag proteins or proteins with
natural metal binding groups by means of metal chelate affinity precipitation. Optimization of the polymer composition and
the type of chelating ions allows for target protein yields of 80% and higher.
| Original language | English |
|---|---|
| Pages (from-to) | 213-220 |
| Journal | Nature Protocols |
| Volume | 2 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2007 |
Subject classification (UKÄ)
- Industrial Biotechnology