2026-09-19T07:25:58Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/987872026-06-16T18:55:03Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022
Alice Y. Ting.
Martell, Jeffrey Daniel
Massachusetts Institute of Technology. Department of Chemistry.
Massachusetts Institute of Technology. Department of Chemistry
2015-09-17T19:12:06Z
2015-09-17T19:12:06Z
2015
2015
http://hdl.handle.net/1721.1/98787
921142287
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2015.
Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Proteins perform myriad functions inside cells. In vitro studies of purified proteins yield important information about their function, but it is an important pursuit in cell biology to study proteins within the native context of intact cells, since their physiological functions might not be recapitulated in any other environment. The most basic question, the location of a protein within the cell, is difficult to answer, since most proteins do not possess a spectroscopic handle that causes them to stand out above all the other proteins and biomolecules in the cell. Fluorescent proteins such as GFP have been revolutionary in allowing specific proteins to be tracked within living cells, but the resolution of fluorescence microscopy (~200 nm) is often inadequate for determining precise subcellular localization. Electron microscopy (EM), on the other hand, offers far superior resolution (<10 nm), but no genetic tag equivalent to GFP has been developed for EM. Horseradish peroxidase (HRP), a multifunctional reporter enzyme widely used in bioanalytical assays, has been utilized for EM because of its ability to generate electron-dense deposits. In addition, HRP produces extremely sensitive chemiluminescent, colorimetric, and fluorescent readouts and has applications for promiscuous protein labeling and detection of hydrogen peroxide. However, the failure of HRP to become active in most cellular compartments has hampered its adoption as a genetic tag. This thesis describes our engineering of genetically encoded peroxidase probes with diverse applications in cell biology. We describe the development of enhanced ascorbate peroxidase (APEX), a HRP-like tag that works in all cellular compartments. We present our applications of this tag for EM imaging, live-cell proteomic mapping, and intracellular detection of hydrogen peroxide. We also discuss the discovery and characterization of APEX2, a greatly improved variant of APEX. Finally, we describe the development of a split HRP protein complementation assay using a combination of rational design and directed evolution, followed by our application of split HRP for detection of neurological synapses.
by Jeffrey Daniel Martell.
Ph.D.
288 pages
eng
Massachusetts Institute of Technology
M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.
http://dspace.mit.edu/handle/1721.1/7582
Chemistry.
Engineered heme peroxidases as genetically encoded probes with diverse applications in cell biology
Thesis
application/pdf
Publication
unknown
unknown
<Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="4937c7b6-26dc-4956-b748-e33903283b7a">
<Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type>
<Language>eng</Language>
<Title>Engineered heme peroxidases as genetically encoded probes with diverse applications in cell biology</Title>
<PublishedIn>
<Publication>
</Publication>
</PublishedIn>
<PublicationDate>2015</PublicationDate>
<Authors>
<Author>
<DisplayName>Martell, Jeffrey Daniel</DisplayName>
<Affiliation>
<OrgUnit>
</OrgUnit>
</Affiliation>
</Author>
</Authors>
<Editors>
</Editors>
<Publishers>
<Publisher>
<DisplayName>Massachusetts Institute of Technology</DisplayName>
<OrgUnit />
</Publisher>
</Publishers>
<License>http://dspace.mit.edu/handle/1721.1/7582</License>
<Keyword>Chemistry.</Keyword>
<Abstract>Proteins perform myriad functions inside cells. In vitro studies of purified proteins yield important information about their function, but it is an important pursuit in cell biology to study proteins within the native context of intact cells, since their physiological functions might not be recapitulated in any other environment. The most basic question, the location of a protein within the cell, is difficult to answer, since most proteins do not possess a spectroscopic handle that causes them to stand out above all the other proteins and biomolecules in the cell. Fluorescent proteins such as GFP have been revolutionary in allowing specific proteins to be tracked within living cells, but the resolution of fluorescence microscopy (~200 nm) is often inadequate for determining precise subcellular localization. Electron microscopy (EM), on the other hand, offers far superior resolution (<10 nm), but no genetic tag equivalent to GFP has been developed for EM. Horseradish peroxidase (HRP), a multifunctional reporter enzyme widely used in bioanalytical assays, has been utilized for EM because of its ability to generate electron-dense deposits. In addition, HRP produces extremely sensitive chemiluminescent, colorimetric, and fluorescent readouts and has applications for promiscuous protein labeling and detection of hydrogen peroxide. However, the failure of HRP to become active in most cellular compartments has hampered its adoption as a genetic tag. This thesis describes our engineering of genetically encoded peroxidase probes with diverse applications in cell biology. We describe the development of enhanced ascorbate peroxidase (APEX), a HRP-like tag that works in all cellular compartments. We present our applications of this tag for EM imaging, live-cell proteomic mapping, and intracellular detection of hydrogen peroxide. We also discuss the discovery and characterization of APEX2, a greatly improved variant of APEX. Finally, we describe the development of a split HRP protein complementation assay using a combination of rational design and directed evolution, followed by our application of split HRP for detection of neurological synapses.</Abstract>
<Access xmlns="http://purl.org/coar/access_right"
>
</Access>
</Publication>