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A new method for the orthogonal labeling and purification of Toxoplasma gondii proteins while inside of the host cell

Wier, Gregory M. (2015) A new method for the orthogonal labeling and purification of Toxoplasma gondii proteins while inside of the host cell. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Toxoplasma gondii is an obligate intracellular protozoan parasite that is estimated to infect a third of the human population. While parasite infection is usually asymptomatic in healthy individuals, it is responsible for life threatening disease in individuals with compromised immune systems. The obligate intracellular nature of the parasite makes its ability to successfully invade and interact with its host cell of paramount importance. How T. gondii interacts with its host cell to permit its survival and replication is still largely understood. Our knowledge of host-pathogen interaction would be strengthened if a system were available to specifically label parasite proteins in the context of an infected host cell. My thesis work in the Boyle laboratory has focused on developing such a system in T. gondii.
I have created a strain of T. gondii that expresses a mutant form of a bacterial methionyl-tRNA synthetase (MetRSNLL), which permits methionine (Met) tRNA to be charged with the azide-containing Met analog, azidonorleucine (Anl). Any protein that incorporates Anl is susceptible to a copper-catalyzed “click” reaction, allowing affinity tags for purification or fluorescent tags for visualization to be appended to them. I show that Anl is only incorporated into parasites expressing the mutant MetRSNLL, and that it is absent from host cell proteins. While the approach allows for the labeling of a large margin of parasite proteins, it appears that N-terminally processed proteins are only minimally labeled. This may be due to the bacterial MetRSNLL only being able to charge Anl to the T. gondii initiator tRNAMet and not the elongator tRNAMet, which would result in only the N-terminal Met residue being replaced with Anl. I have begun work to try to modify the system to permit Anl to be more widely incorporated into parasite proteins. Despite the systems current limitations, it should be useful for studying proteomic changes in the parasite while inside of the host. Furthermore, the system should be readily adaptable for other Apicomplexan parasites, like Plasmodium spp.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Wier, Gregory M.gmw18@pitt.eduGMW18
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairBrodsky, Jeffrey L.jbrodsky@pitt.eduJBRODSKY
Committee MemberPipas, James M.pipas@pitt.eduPIPAS
Committee MemberLawrence, Jeffrey G.jlawrenc@pitt.eduJLAWRENC
Committee MemberBruchez, Marcel
Committee MemberBoyle, Jon P.boylej@pitt.eduBOYLEJ
Date: 23 June 2015
Date Type: Publication
Defense Date: 2 April 2015
Approval Date: 23 June 2015
Submission Date: 22 April 2015
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 93
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Toxoplasma gondii, Click chemistry
Date Deposited: 23 Jun 2015 17:38
Last Modified: 15 Nov 2016 14:27


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