Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers.
Metal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions. The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well-characterized metal affinity macroligands (AML) and their applications to the number of different purification systems. In the present work we describe a detailed study of a new purification system using metal-loaded thermoresponsive copolymers as AML. The copolymers of vinylimidazole (VI) with N-isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol%. When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly expressed histidine-tagged single-chain Fv-antibody fragments (His(6)-scFv fragments) from the fermentation broth free from E. coli cells. Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein-polymer complex in EDTA buffer and subsequent reprecipitation of the polymer. His(6)-scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively. The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer. The SDS-PAGE pattern showed significant purification of the antibody fragments.
Kumar
Ashok
A
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden.
Wahlund
Per-Olof
PO
Kepka
Cecilia
C
Galaev
Igor Yu
IY
Mattiasson
Bo
B
D000180:Acrylic Resins / Q000737:chemistry; D000345:Affinity Labels / Q000032:analysis / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry*; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000302:isolation & purification*; D006358:Hot Temperature; D007093:Imidazoles / Q000737:chemistry*; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000302:isolation & purification*; D008670:Metals / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*
D000180:Acrylic Resins; D000345:Affinity Labels; D002614:Chelating Agents; D007093:Imidazoles; D007128:Immunoglobulin Fragments; D008670:Metals; D011108:Polymers; C055966:immunoglobulin Fv; C052970:poly-N-isopropylacrylamide; D006639:Histidine; D003300:Copper; D009532:Nickel; C425704:N-vinylimidazole