<p>The successful genomic era has resulted in a great demand for efficient production and purification of proteins. The main objective of the work described in this thesis was to develop methods to facilitate recovery of target proteins after recombinant production in Escherichia coli.</p><p>A positively charged purification tag, Z<sub>basic</sub>, has previously been constructed by protein design of a compact three-helix bundle domain, Z. The charged domain was investigated for general use as a fusion partner. All target proteins investigated could be selectively captured by ion-exchange chromatography under conditions excluding adsorption of the majority of Escherichia coli host proteins. A single cation-exchange chromatography step at physiological pH was sufficient to provide Z<sub>basic</sub> fusion proteins of high purity close to homogeneity. Moreover, efficient isolation directly from unclarified <i>Escherichia coli</i> homogenates could also be accomplished using an expanded bed mode. Since the intended use of a recombinant protein sometimes requires removal of the purification tag, a strategy for efficient release of the Z<sub>basic</sub> moiety using an immobilised protease was developed. The protease columns were reusable without any measurable decrease in activity. Moreover, subsequent removal of the released tag, Z<sub>basic</sub>, was effected by adsorption to a second cation-exchanger. </p><p>Using a similar strategy, a purification tag with a negatively charged surface, denoted Z<sub>acid</sub>, was constructed and thoroughly characterised. Contrary to Z<sub>basic</sub>, the negatively charged Z<sub>acid</sub> was highly unstructured in a low conductivity environment. Despite this, all Z<sub>acid</sub> fusion proteins investigated could be efficiently purified from whole cell lysates using anion-exchange chromatography</p><p>Synthesis of polypeptides occurs readily in Escherichia coli providing large amounts of protein in cells of this type, albeit often one finds the recombinant proteins sequestered in inclusion bodies. Therefore, a high throughput method for screening of protein expression was developed. Levels of both soluble and precipitated protein could simultaneously be assessed <i>in vivo</i> by the use of a flow cytometer. </p><p>The positively charged domain, Z<sub>basic</sub>, was shown also to be selective under denaturing conditions, providing the possibility to purify proteins solubilised from inclusion bodies. Finally, a flexible process for solid-phase refolding was developed, using Z<sub>basic</sub> as a reversible linker to the cation-exchanger resin.</p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:kth-471 |
| Date | January 2005 |
| Creators | Hedhammar, My |
| Publisher | KTH, School of Biotechnology (BIO) |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, text |
Page generated in 0.0018 seconds