| 2. |
- Kumar, Ashok, et al.
(författare)
-
Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers
- 2003
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 84:4, s. 494-503
-
Tidskriftsartikel (refereegranskat)abstract
- 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 (His6-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. His6-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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 494-503, 2003.
|
|
| 3. |
- Wahlund, Per-Olof, et al.
(författare)
-
"Protein-like" copolymers: Effect of polymer architecture on the performance in bioseparation process
- 2002
-
Ingår i: Macromolecular Bioscience. - 1616-5195. ; 2:1, s. 33-42
-
Tidskriftsartikel (refereegranskat)abstract
- Recently, a new class of copolymers, so-called protein-like copolymers has been predicted theoretically by computer simulation. In these copolymers. the conformation of the copolymer determines the exposure of certain comonomer units to the outer solution. Depending on the conformation, copolymer molecules with essentially the same comonomer composition could have pronouncedly different properties. The authors demonstrated experimentally such behavior in case of poly[(N- vinylcaprolactam)-co-(N-vinylimidazole)] (Dokl. Chem. 2001,375, 637). One more group of copolymers with protein-like behavior is copolymers of N-isopropylacryl-amide with N-vinylimidazole. Poly[(N-isopropylacryl-amide)-co-(N-vinylimidazole)] was synthesized by radical polymerization and separated into two fractions using immobilized metal affinity chromatography on Cu2+-loaded iminodiacetic acid Sepbarose CL 6B (Cu2+-IDA-sepharose). The unbound fraction which passed through the column and bound fraction eluted with ethylenediaminetetraacetic acid, disodium salt (EDTA) solution differed significantly in molecular weight, 1.4 x 10(6) and 1.35 x 10(5), respectively but were very close in comonomer composition, 7.8 and 9.1 mol-% of imidazole, respectively. The composition of bound fraction was confirmed by titration of imidazole groups. Despite close chemical composition, the bound and unbound fraction behaved differently with respect to temperature-induced phase separation at different pH values, the dependence of hydrodynamic diameter on pH and concentration of Cu2+- ions, and the coprecipitation of soybean trypsin inhibitor with the copolymer in the presence of Cu2+-ions. The differences in the behavior of copolymer fractions are rationalized assuming that the bound fraction presents a protein-like copolymer. The dependence of hydrodynamic diameter of bound (closed symbols) and unbound (open symbols) poly(NI-PAAM-VI) at different Cu2+/vinylimidazole ratios (n(Ca)/n(VI)), The polymer concentration was 4.5 mg (.) ml(-1) and pH 7.5 was obtained in all systems by using 0.010 m HEPES as a buffer. Mean values from five correlation functions are given.
|
|
