Producing Glycosaminoglycan-Binding Proteins Containing Single-Isotope-Labeled Lysine as a Tool to Study Protein-Glycosaminoglycan Interactions

Pleiotrophin (PTN) is a cell-signaling protein in the human body that plays a pivotal role in the development of the central nervous system. It is known to have a high affinity for glycosaminoglycan (GAG), a type of linear polysaccharide. PTN has the ability to bind to a wide range of receptors, including receptor-type protein tyrosine phosphatase ζ (PTPRZ), a protein expressed in embryonic stem cells that regulates signals associated with survival, cell proliferation, and stem cell pluripotency. Several of these receptors are proteoglycans that carry GAGs, and the interaction between PTN and GAG has proven to be crucial to PTN’s functionality. Though PTN performs several important biochemical duties in normal cellular processes, this protein is upregulated in various cancer cell lines, primarily glioblastoma, an aggressive form of cancer that arises in the brain or spinal cord. The high levels of PTN expression in these forms of cancer may correlate to the cancer cells’ metastatic ability in the body. Determining how these PTN-GAG interactions form in cells is imperative for understanding how they may correlate to the development of cancer cell lines such as glioblastoma. However, due to the NMR signal degeneracy among the lysines in PTN, it is currently not possible to distinguish between lysines that have strong interactions with GAG and those that do not. To overcome this, pyrrolysyl-tRNA synthetase-mediated amber codon suppression is used to incorporate a single 15N-labeled lysine, Boc-lysine (Boc-K), at a specific position. This thesis seeks to optimize the systems and conditions needed to achieve amber codon suppression. The Origami B (DE3) strain is commonly used to achieve this, and demonstrates positive expression of PTN. The first aim of this project is to determine whether SHuffle® demonstrates enhanced expression of PTN and, therefore, incorporation of Boc-K. However, upon comparing PTN expression results, it was found that SHuffle® and Origami B(DE3) demonstrated similar levels of PTN expression. This project's second phase is focused on using C321.ΔA (Church) strain to evaluate differences in PTN expression compared to SHuffle® and Origami B(DE3). Expression testing indicated, however, that the expression of PTN in Church strain was inconclusive.