title: Efficient expression and purification of human aglycosylated Fcgamma receptors in Escherichia coli.
abstract: Effector Fc gamma receptors (FcgammaRs) are expressed on the surface of a variety of cells of hematopoietic lineage and serve as a bridge between adaptive and innate immune responses. The interaction between immune complexes, formed by IgG class antibodies that are crosslinked with antigen, and FcgammaRs triggers signaling cascades that result in numerous cellular responses including the activation or donwregulation of cytotoxic responses, cytokine release, and antibody synthesis. Here, the extracellular domains of the human type I transmembrane FcgammaRs were expressed in Escherichia coli and their interactions to subclass IgGs (IgG1, IgG2, IgG3, and IgG4) antibodies were analyzed. Expression using fully synthetic E. coli codon optimized FcgammaR genes and optimization of sequences for N-terminal translation initiation region through mRNA secondary structure prediction enabled us to achieve high yield of purified, bacterially expressed receptors, including FcgammaRI and FcgammaRIIIa which have not been successfully expressed in bacteria until now. The aglycosylated FcgammaRs showed similar IgG subclass binding selectivity compared to the respective glycosylated FcgammaRs expressed in mammalian cells.
authors.affiliation: Department of Chemical Engineering, University of Texas, Austin, 78712, USA.
authors.forename: Sang Taek
authors.initials: ST
authors.lastname: Jung
authors.forename: Tae Hyun
authors.initials: TH
authors.lastname: Kang
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D004926:Escherichia coli / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D017452:Receptors, IgG / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D017452:Receptors, IgG; D011994:Recombinant Proteins