Optimization of ELP-Intein Protein Purification System By Comparing Four Different Self-Cleavage ELP Fusion Proteins

Abstract

<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>