Engineering a light-activated protein domain for the site-specific conjugation of native IgGs / James Zhe Hui.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

Hui, James Zhe, author.

Contributor:

Tsourkas, Andrew, degree supervisor.

Diamond, Scott, degree committee member.

Greene, Mark, degree committee member.

Luning Prak, Eline, degree committee member.

University of Pennsylvania. Department of Bioengineering, degree granting institution.

Language:

English

Subjects (All):

Penn dissertations--Bioengineering.

Bioengineering--Penn dissertations.

Local Subjects:

Penn dissertations--Bioengineering.

Bioengineering--Penn dissertations.

Physical Description:

viii, 167 leaves : illustrations; 29 cm

Production:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2015.

Summary:

Antibody conjugates have been used in a variety of applications from immunoassays to targeted therapies. However, the existing conjugation methods suffer from a wide array of drawbacks, ranging from the production of heterogeneous or partially inactivated products when using chemical methods, to the requirements for long reactions times when using enzymatic methods. What's more, it is increasingly clear that in order to maximize an antibody's antigen binding and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto the Fc region of the IgG site-specifically. We have developed a facile method, termed LASIC (Light Activated SIte-specific Conjugation) for the site-specific modification of full length, native IgGs by using engineered Protein Z and Protein G adapter domains that can form covalent links to the Fc regions upon exposure to non-harmful long wavelength (365nm) UV light. By combining unnatural amino-acid incorporation and expressed protein ligation (EPL) techniques, we not only can produce these the adapters efficiently using the E. coli expression system, but also easily introduce a variety of functional moieties such as azides, fluorophores or biotin handles, carried on short synthetic peptides, into their C-terminals. Using these adapters, we have site-specifically labeled a range of native IgGs with a number of reactive groups. We have also demonstrated LASIC's utilities in functionalizing nanoparticles with IgGs. Unlike alternative site-specific conjugation techniques that are often only compatible with some IgGs and/or require lengthy cloning or harsh chemical/enzymatic treatments, the LASIC method is compatible with nearly all IgGs from various host and does not damage the IgG due to its use of long wavelength UV light. Labeling occurs with unprecedented efficiency and speed (>90% after 30 min) and with no impact on IgG affinity. Taken together, this technique represents a vastly more accessible and efficient approach for conjugating "off-the-shelf" IgGs, thus making site-specific conjugation more available to many at large.

Notes:

Ph. D. University of Pennsylvania 2015.

Department: Bioengineering.

Supervisor: Andrew Tsourkas.

Includes bibliographical references.

OCLC:

950747203