Expression and purification of the predicted PEG10 aspartyl protease domain
Paternally Expressed Gene 10 (PEG10) is an imprinted, retrotransposon-derived gene found in mammals. Although many of the retrotransposon domains have become degenerated in PEG10, a predicted retroviral-type aspartyl protease (AP) domain has been highly conserved. Retroviral-type APs play a crucial role in the replication of some retroviruses such as the Human Immunodeficiency Virus (HIV) and are there fore important drug targets. Consequently, extensive biochemical and structural data are available for this class of proteins, although the vast majority of this has been gathered from only a small number of retroviral enzymes. Preliminary evidence indicates that the PEG10 AP is an active protease, although proteolysis by this enzyme has yet to be observed in vitro (Clark et al., 2007). This study aimed to express, purify, and characterise the predicted PEG10 AP.A number of PEG10 AP clones, each with different termini and across more than one recombinant expression system, were expressed to produce the PEG10 AP domain in E. coli. The majority of expressed proteins were largely insoluble and unsuitable for further characterisation. One clone, however, produced soluble PEG10 AP in sufficient quantities for purification and further analysis. Several lines of evidence indicated that the purified protein was dimeric in solution, consistent with the quaternary structure of other retroviral-type APs.The results presented in this thesis support the hypothesis that the PEG10 AP is active and has retained characteristics from the ancestral retrotransposon enzyme. The expression and purification protocol that has been developed can now be used to generate PEG10 AP for detailed biophysical and functional characterisation. Widely used protease inhibitors used for treatment of infection with HIV have pleiotropic effects in patients and are known to inhibit a diverse range of proteases. The development of an in vitro activity assay and testing whether the PEG10 AP is a serendipitous target of these inhibiting compounds will not only enhance our understanding of retroviral-type APs but potentially contribute to their use as therapeutic agents.
Advisor: Poole, Elizabeth; Tate, Warren
Degree Name: Master of Science
Degree Discipline: Department of Biochemistry
Publisher: University of Otago
Keywords: retropepsin; protease; aspartyl protease; retroviral protease; LTR-retrotransposon; PEG10
Research Type: Thesis