Specific Precipitation of Biomolecules Using Synthetic Polymers

Detta är en avhandling från Per-Olof Wahlund, Dept. of Biotechnology, Lund University

Sammanfattning: Purification is a very important stage in biotechnological processes. The traditionally used precipitation techniques are generally nonspecific and thus one or more chromatographic steps are normally required. There is thus a need for new approaches to improve existing techniques. New, more specific precipitation techniques have been introduced by the use of stimuli-responsive polymers. These polymers are useful in bioseparation due to their ability to form a separate phase following a slight change in conditions. This thesis describes the use of two types of polymer systems for specific precipitation of biomolecules; a thermo-responsive polymer and the formation of polyelectrolyte complexes (PECs). Affinity precipitation of a His-tagged antibody fragment was performed by metal chelate affinity precipitation using Cu(II) or Ni(II) bound to the copolymer of N-isopropylacrylamide and N-vinylimidazole (poly(NIPAAM-VI)). The antibody fragment was purified with a yield of 80-90% and a purification fold of 16-20. Metal chelate affinity precipitation is thus a simple and efficient technique for the purification of proteins with an affinity for metal ions. The scale-up of this technique is also expected to be straightforward. The introduction of the relatively hydrophilic VI groups into poly(NIPAAM) together with the addition of metal ions significantly affects the precipitation behaviour. In this work it was also found that the architecture of copolymer chains of poly(NIPAAM) is important for the polymer behaviour and thus determines the usefulness of the polymer in various applications. The formation of polyelectrolyte complexes between nucleic acids and synthetic polycations was studied. The factors found to control phase separation in the PECs were: charge ratio, salt concentration, chain length of the polycation, chain length of the nucleic acid and the affinity of counterions for the polyelectrolytes. PECs including nucleic acids follow the same general rules as those for PECs consisting of synthetic polyanions and polycations. The special properties of the double helix of DNA appear to be responsible for the difference in phase separation behaviour. Compared to synthetic polyanions, DNA is more prone to forming insoluble PECs, and an extension in the region of insoluble PECs is obtained in the phase diagram. On the other hand, DNA forms much more stable complexes than RNA. This finding provided the basis for the development of a step for the separation of plasmid DNA from a clarified alkaline lysate. The plasmid recovery was close to 80% and more than 90% of the RNA and 90% of the proteins were removed.

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