Simple salts are known to influence the cloud point temperature of the aqueous solution of thermoresponsive compounds, such as poly-(N-isopropylacrylamide), PNIPAM. The effect of one series of anions (six potassium salts) and two series of cations (five alkali-metal hydroxides and chlorides, respectively) on the cloud point temperature of PNIPAM oligomers is investigated. All salts with the exception of low (<0.5 M) concentrations of KI are found to lower the cloud point temperature; the relationship between the temperature and the concentration of the added salt tends to be linear. The intensity of the effect of a given salt corresponds to its position in the Hoffmeister series. However, while the anions differ significantly in force, the cations-with the exception of Li + -show little difference in this regard. The observed effects are interpreted based on the structure making/structure breaking potential of the involved ions as evidenced by their viscosity B coefficients. While a relationship could be established for the anions this is less obvious for the cations. Moreover, while the structure breaking ability of the I --anion to some extent explains its salting in ability at low concentrations, it does not account for the linear salting out effect observed at higher concentrations. The second attempt to interpret the results is based on the solvophobic theory, i.e., takes the contribution of both electrostatic and hydrophobic interactions into account. Thermodynamic data on the phase separation obtained through differential scanning calorimetry are used to calculate the unitary free Gibbs energy as a function of the molal salt concentration.
Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triple-helix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT)(7)), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive N-isopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4 degrees C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40 degrees C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70% and 90%. Non-specific co- precipitation of either the target molecule by the non-activated AML precursor or of contaminants by the AML were below 7% and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.
Affinity precipitation, especially secondary effect affinity precipitation, has repeatedly been suggested as a valuable technique for the biotechnical downstream process. The present lack of applications is related to the scarcity of predictable affinity macroligands and to the fact that rather high affinity constants are required in affinity precipitation (K(D) < 10(-10)). The latter are rarely found in nature, at least in the case of small affinity ligands (affinity tags), and are usually difficult to handle (complex dissociation) once one has found them. In this article we describe a new type of thermoresponsive affinity macroligand. The base polymer (poly-N-isopropylacrylamide, or PNIPAAm) is produced by chain transfer polymerization. As a consequence, the structure, as well as the solubility behavior, is very homogeneous (polydispersity < 1.2), whereas the average molecular mass is small (<5000 g/mol). In pure water, the base polymer shows sharp thermoprecipitation at 32.2 degrees C. Each oligomer carries a single amino end group, which allows easy and defined coupling of the affinity ligand, while preserving the ligand's activity to the highest possible degree. Herein, the oligomer was coupled to iminobiotin. The ensuing affinity macroligand has a high affinity to avidin (and avidin-tagged molecules) at elevated pH (<10), but releases the avidin easily at lower pH (approximately 4). The affinity macroligands were used to purify avidin from solutions containing large amounts of lysozyme as well as from cell culture supernatants containing 5% fetal calf serum. In both cases, pure avidin was recovered (residual protein contamination below the detection limit), with yields of >90%.
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