SIR Medicine Investigation Abstract
CLONING AND EXPRESSION OF HUMAN FC RECEPTOR LIGAND BINDING DOMAINS
Presenter:
Edward Nepomuceno, Illinois Mathematics and Science Academy, 1500 W. Sullivan Road, Aurora, IL 60506
Mentor:
Dr. Barry Arnason, University of Chicago
Abstract:
Immune complexes (ICs), antibodies bound to cognate antigens, serve as key regulators of the immune system by acting as liaisons between the adaptive and innate immune responses. Varying in size and structure, ICs bind preferentially to low-affinity Fc receptors (FcRs) which in humans are found to be Fc?RIIa, Fc?RIIb, Fc?RIIc, Fc?RIIIa, and Fc?RIIIb. Growing evidence indicates that larger ICs preferentially bind Fc?RIIIa (activatory receptor) as opposed to small ICs which interact predominantly with Fc?RIIb (inhibitory receptor). The cDNAs for Fc?RIIa and Fc?RIIb were isolated from the cell lines K562 and U937 RNA respectively. Using splice-overlap extension, the ligand-binding domains of each receptor were subcloned, and a 6xHis tag was added to the sequences of interest for the purpose of single-step purification via immobilized metal affinity chromatography (IMAC). The LBD-6xHis cDNAs were transferred into the expression vector, pcDNA3.0. Chinese hamster ovary cells (CHO cells) were then used to express the FcR LBDs. The overall goal is to use the low-affinity FcRs, expressed as soluble proteins, to study LBD-Ligand interactions using techniques such as immunoprecipitation and ELISA. The yield from these studies will be a clearer understanding of the relationship between IC size and structure and its immunoregulatory properties.