Protein Semi-Synthesis in Vivo

Author

Izabela Giriat Stankiewicz

Date of Award

2003

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Muir Laboratory

Abstract

Incorporation of chemical probes into proteins is a powerful way to elucidate biological processes and to engineer novel function. This thesis describes the ligation of synthetic molecules to target proteins in an intracellular environment, and the use of semi-synthetic proteins for whole animal studies. In the first approach a cellular protein is genetically tagged with one half of a split-intein. The complementary half is linked in vitro to the synthetic probe and this fusion is delivered into cells using a transduction peptide. Association of the intein halves in the cytosol triggers protein transsplicing, resulting in the ligation of the probe to the target protein through a peptide bond. This process is specific and applicable to cytosolic and integral membrane proteins. The technology should allow cellular proteins to be elaborated with a variety of abiotic probes. In the second approach a sequential expressed protein ligation was used to link three fragments: a targeting protein, an affinity tag, and an imaging moiety. Specifically, vascular endothelial growth factor (VEGF) was ligated with a 6xHis tag and a synthetic high affinity ^99mTc chelator. Following protein semi-synthesis, the ^99mTc was incorporated into the chelator and the ^99mTC-labeled protein was injected into mice bearing a tumor. The semi-synthetic VEGF localized onto the vascularized tumor and allowed it to be imaged with a γ camera. The strategy developed is modular, permitting the use of multiple chemical moieties, purification methods, and targeting proteins. This methodology could be used to provide insights into biological processes in normal development and homeostasis, as well as during pathogenic events such as cancers.

Comments

A thesis submitted to the faculty of the Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Recommended Citation

Stankiewicz, Izabela Giriat, "Protein Semi-Synthesis in Vivo" (2003). Student Theses and Dissertations. 374.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/374