title: Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli.
abstract: Human FcγRI (CD64) is an integral membrane glycoprotein functioning as a high-affinity receptor binding to monomeric IgG. In this study, the extracellular region of FcγRI, which is the actual part that interacts with IgG, was expressed as aglycosylated recombinant human FcγRI (rhFcγRI) in Escherichia coli. The soluble form of aglycosylated rhFcγRI was expressed in the periplasm of E. coli. The production of soluble aglycosylated rhFcγRI was increased by low induction levels. Furthermore, this production was increased by low translational efficiency, controlled by modification of the putative region between the ribosome binding site and initiation codon of rhFcγRI fusing signal peptide (MalE, PelB, or TorT) of the expression vector. By the optimization of induction and translational efficiency, the production of soluble aglycosylated rhFcγRI was up to approximately 0.8 mg/l of culture medium. Surface plasmon resonance analysis revealed that the binding affinities of aglycosylated rhFcγRI for human IgG1 (equilibrium dissociation constant K D =[1.7±0.2]×10−10 M) and IgG3 (K D=[1.1±0.2]×10−10 M) were similar to those of glycosylated rhFcγRI.
authors.affiliation: Sagami Chemical Research Institute, Ayase, Kanagawa, Japan. hatayama@sagami.or.jp
authors.forename: Kouta
authors.initials: K
authors.lastname: Hatayama
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Asaoka
authors.forename: Megumi
authors.initials: M
authors.lastname: Hoya
authors.forename: Teruhiko
authors.initials: T
authors.lastname: Ide
mesh_terms: D003062:Codon; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D019897:Periplasm / Q000737:chemistry; D014176:Protein Biosynthesis; D017452:Receptors, IgG / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003062:Codon; D017452:Receptors, IgG; D011994:Recombinant Proteins