<p> Proteins vary tremendously in many of their physical and chemical properties.
In order to perform in vitro application or analysis, one protein must be separated
from other cellular components. This process is called protein purification. With the
advances of modem science and technology, many protein purification schemes have
been developed. Among them, the ELP-intein protein purification system has recently
attracted an increasing amount of attention because of its positive characteristics: it is
simple, inexpensive, scalable, with a high throughput, protease-free, etc. However,
although the scientific literature reports all those good aspects of the system, several
bad responses to it still exist. In this thesis, through comparing expression and
purification of four different self-cleavage ELP fusion proteins, we propose a general
solution to these problems for the first time. This makes a significant contribution to
increased utility of the method of protein purification using self-cleavable stimulus
responsive tags. </p> <p> When ELP-intein fusion proteins are expressed in bacteria, formation of non-native cytoplasmic aggregates (inclusion bodies) is a common problem which
affects the yield of target protein. Inclusion bodies are generally assumed to contain
misfolded or partially folded protein through exposure of hydrophobic patches and the
consequent intermolecular interactions. Despite a loss of total expression yield and the
need for more time, culturing at a lower temperature was reported to promote the
expression of genes into soluble proteins and alleviate IB formation. Directly motivated by previous reports, we have applied a low-temperature expression strategy
to solve the problems in this research. As expected, most of the T4-ELP inclusion
bodies disappeared, and were transformed into a soluble expression, when culturing at
lower temperatures. </p> <p> Inverse transition cycling (lTC), as the core method for the system we investigated has proved successful in the past with proteins that were expressed to high levels. However poor level ELP-intein tagged protein expression happens from time to time. It is hypothesized that if an ELP tagged molecule is present in a solution at a very low
concentration, adding an excess amount of free ELP to the sample would form hybrid
aggregates via the interaction of ELP moieties of the two molecules. We used this
efficient and reversible capture system for low yield recombinant protein purification,
and found it is perform very well. </p> / Thesis / Master of Applied Science (MASc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22380 |
| Date | 08 1900 |
| Creators | Liu, Han |
| Contributors | Filipe, Carlos, Chemical Engineering |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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