Construction, expression, and purification of soluble CD16 in bacteria
Sinotte, Christopher Matthew
MetadataShow full item record
CD16 is a physiologically essential Fc and #947; receptor III as either a single- pass transmembrane protein (CD16A) or as a glycosylated phosphatidylinositol (GPI) anchored protein (CD16B) on the surface of immune cells that have been implicated in many autoimmune and immune complex-mediated diseases. Its functions include binding and clearing antibody (IgG) coated foreign pathogens, receptor-mediated phagocytosis, and triggering antibody dependent cellular cytotoxicity. It is well established that these functions depend on protein-protein interaction between CD16 and the Fc domain of IgG. However, the molecular details of CD16-IgG interactions are less well defined, but are essential to developing therapeutic compounds to treat many autoimmune and IC diseases. Stable mammalian cell lines expressing wild-type CD16 isoforms and site-specific mutants, including extracellular soluble fragments of CD16 have been established. Soluble forms of wild type CD16A and these CD16 mutants were expressed in a bacterial pathway in order to amass sufficient quantities for x-ray crystallographic studies. The soluble portions of wild-type CD16A and several site-specific CD16A and CD16B mutants were constructed by PCR amplification and ligation with a pET vector. The proteins were expressed in a prokaryotic pathway, BL21 AI, for 8-10 hours and lysed to obtain inclusion bodies. A hand-held sonicator was used to wash the inclusion bodies, while a Urea solution separated and dissolved the proteins. The target proteins were then refolded by rapid dilution, concentrated with a stir cell, and purified. Wild type sCD16A and four site specific mutants were constructed with good sequencing, while wild type sCD16A, sCD16A F176V, and sCD16A G147D were expressed and refolded to optimal levels. X-ray crystallographic data has been collected from sCD16A F176V as a result of these studies and crystals are currently being grown from wild type sCD16A and sCD16A G147D.