Purification, characterization and binding of metals to Zinc Alpha 2-Glycoprotein (ZAG) by employing various techniques

Abstract

In this thesis ―Purification, Characterization and Binding of Metals to Zinc Alpha 2-Glycoprotein (ZAG) by Employing Various Techniques‖ the development of several techniques were carried out to analyze the interaction of biologically important metals of Irving William Series. The expression and purification of the ZAG protein was also optimized for the preparation of samples. The folding to the native conformation was also carried out. That ZAG protein is present in almost all fluids of the body. Since the discovery of ZAG, several studies have been carried out. The importance of ZAG as an important biochemical agent is known long before. The growing number of literature since today demonstrates that it’s an important biomarker for the cancer cachexia, prostate cancer, lipid mobilizing agent and obesity suppressor. The crystal structure of ZAG reveals that the fold and distribution of domains are similar to the Major histocompatibility complex-I (MHC-I) molecules. This class is known for their immune regulation and thus ZAG is believed as a central player in the immune system and cellular signaling. There is no clear evidence of ZAG participation in the cancerous conditions but over expression is dealt as a biomarker. The study shows that the ZAG interacts with several ligands. There interaction with metals is known for boosting the bioactivity of ZAG. Although, it’s believed long ago that the ZAG is an important protein that participate in several biochemical processes and little is known to understand the ligands bound conformations of ZAG with their targets like peptides and metals. In this current project, we were focused to get the good expression and purification procedure for the preparation of ZAG. The unnative aggregated fold of ZAG from the inclusion bodies was transformed into the native fold using refolding and dialysis method optimization. The introduction of histidine tag to the C-terminal and their successful removal by the Carboxypeptidase Y enzyme was also carried out. The results of the cleavage of histidine tag were evaluated with gel electrophoresis, Mass Spectrometry and Fluorescence Spectroscopy. The confirmation of native fold and bioactivity was also confirmed from DAUDA binding affinity using fluorescence emission. The formation of metal complex of Irving William Series with ZAG and their respective complexation analysis was also studies using MALDI-TOF MS and Fluorescence Spectroscopy. xiii It was found that the ZAG has multiple binding sites that interact with metals even in the presence of DAUDA. The X-ray tube analysis using FPXRF shows that Fe (III) and Co (II) has similar concentration of atoms bound to ZAG molecules.