Metal-chelate affinity precipitation of proteins using responsive polymers.
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.
Mattiasson
Bo
B
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, SE-22100 Lund, Sweden. Bo.Mattiasson@biotek.lu.se
Kumar
Ashok
A
Ivanov
Alexander E
AE
Galaev
Igor Y
IY
D000178:Acrylamides / Q000138:chemical synthesis* / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D011232:Chemical Precipitation; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000138:chemical synthesis* / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D011108:Polymers / Q000138:chemical synthesis* / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D013696:Temperature
D000178:Acrylamides; D002614:Chelating Agents; D007093:Imidazoles; D008670:Metals; D011108:Polymers; D011506:Proteins; C067295:N-isopropylacrylamide; C425704:N-vinylimidazole