title: Engineering of recombinant human Fcγ receptor I by directed evolution.
abstract: Human FcγRI is a high-affinity receptor for human IgG. On the basis of its binding activity, recombinant human FcγRI (rhFcγRI) has several possible applications, including as a therapeutic reagent to treat immune complex-mediated disease and as a ligand in affinity chromatography for purification of human IgG. As the stability and production rate of rhFcγRI are low, it would need to be engineered for use in such applications. In this study, we demonstrated engineering of rhFcγRI by directed evolution through random mutagenesis and integration of mutations. Engineered rhFcγRI was expressed by Escherichia coli. Screening identified 19 amino acid mutations contributing to the thermal stability and production rate of rhFcγRI. By integration of these mutations, engineered rhFcγRI containing all 19 amino acid mutations (enFcRd) was constructed and showed markedly enhanced thermal stability (transition midpoint temperature [Tm] = 65.6°C) and production rate (3.27 mg L-medium(-1) OD(600)(-1)) compared with wild-type rhFcγRI (Tm = 48.5°C; production rate, 0.07 mg L-medium(-1) OD(600)(-1)) without a change in the specificities of binding to human IgG subclasses. Moreover, the binding affinity of enFcRd for human IgG1/к (equilibrium dissociation constant [K(D)] = 0.80 × 10(-10) M) was higher than that of wild-type rhFcγRI (K(D) = 1.23 × 10(-10) M). Our study showed that substantial engineering of rhFcγRI is possible.
authors.affiliation: Tosoh Corporation, Ayase, Kanagawa, Japan.
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Asaoka
authors.forename: Kouta
authors.initials: K
authors.lastname: Hatayama
authors.forename: Kanta
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tomita
authors.forename: Teruhiko
authors.initials: T
authors.lastname: Ide
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D055550:Protein Stability; D017452:Receptors, IgG / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D017452:Receptors, IgG; D011994:Recombinant Proteins