Elastin-like polypeptides (ELP) are artificially designed protein biopolymers that can be produced by living organisms. These proteins have the unique ability to undergo reversible inverse phase transition, in response to changes in temperature and/or addition of chaotropic salts. Below the transition temperature (T1) , ELP is soluble in water. Increasing the temperature above Ti, ELP coacervates into an aqeous ELP-rich phase. In this thesis, this unique feature of ELP was used in for recombinant protein purification and for the formation of aqueous multiple-phase systems. For protein purification, ELP was fused with an intein and a model protein (thioredoxin), to demonstrate a simple and inexpensive approach for recombinant protein purification. The ELP tags replace the chromatographic media and the intein replaces the use of the protease in conventional methods. Using ELP tags was found to be consistent with large -scale recombinant protein production/purification by purifying an ELP tagged protein using a stirred cell equipped with a microfiltration membrane. When the
temperature and/or salt concer.tration is increased for mixtures containing free ELP and
ELP tagged proteins, simultaneous phase transition takes place. This served as the basis
for the development of a method suitable for selectively recovering molecules from complex mixtures with high specificity, full reversibility, and virtually unlimited affinity. The second parts of this thesis focus on the ability of ELP to form aqueous twophase systems (A TPS) in vitro and most importantly, in vivo- with the formation of aqueous microcompartments in living cells. These compartments exclude the protein making machinery of the cell, acting as depots for newly expressed protein. It is also shown (in vitro) that ELP bastd droplets exclude proteases, protecting proteins from degradation. These observations are important for high-level production of recombinant proteins. Also described, is the formation of protein based aqueous multiphasic systems, with tunable morphologies. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/16698 |
| Date | 05 1900 |
| Creators | Ge, Xin |
| Contributors | Filipe, Carlos, Chemical Engineering |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | An error occurred on the license name., An error occurred getting the license - uri. |
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