Expression of O-linked N-acetylglucosamine modified proteins and production and characterization of chlamydia trachomatis CT663

• Main Author: Goodwin, Octavia Y
• Other Authors: Warner, Isiah
• Format: Others
• Language: en
• Published: LSU 2014
• Subjects: Chemistry
• Online Access: http://etd.lsu.edu/docs/available/etd-11172014-155433/

O-linked â-N-acetylglucosamine is a regulatory post translational modification. This modification occurs on nearly all functional classes of proteins, in the nucleus and cytoplasm. O-GlcNAc is added to serine or threonine by O-GlcNAc transferase and removed by O-GlcNAcase. Previous attempts to study O-GlcNAc-modified proteins have resulted in low yields, making 3-dimensional structure determination impossible. In this dissertation O-GlcNAc transferase will be co-expressed with domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2) in E. coli, to produce O-GlcNAc-modified protein. The O-GlcNAc-modified protein was expressed in a variety of E. coli cell lines at a variety of conditions, but only small quantities of insoluble protein were produced. A glycosidase was suspected due to the disappearance of the O-GlcNAc modification from the protein. O-(2-acetamido-2-dexoy-dglucopyranosylidene) amino-N-phenylcarbamate (PUGNAc), a â-N-acetylglucosaminidase inhibitor, was added to the culture media and increased the production of O-GlcNAc-modified protein. This was the first evidence that â-N-acetylglucosaminidase (NagZ), an E. coli enzyme, cleaves O-GlcNAc from proteins in vivo. NagZ was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. In E. coli, NagZ cleaves the GlcNAc-â1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm. A NagZ knockout showed no activity towards the O-GlcNAc-peptide, confirming NagZ as the enzyme responsible for cleaving O-GlcNAc from our glycoprotein expressed in vivo. O-GlcNAc-modified protein produced by the NagZ knockout (∆NagZ) co-expression system is highly glycosylated and can be resolubilized from the pellet. The ∆NagZ is a step closer to production of milligram quantities of O-GlcNAc-modified protein for structure determination.