Effective expression of soluble aglycosylated recombinant human Fcγ receptor I by low translational efficiency in Escherichia coli

Hatayama, Kouta; Asaoka, Yoshiharu; Hoya, Megumi; Ide, T.

doi:10.1007/s00253-012-3902-x

Cited by 9 publications

(15 citation statements)

References 24 publications

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“…Overall, they have a modest impact on the ability of the receptor to engage the Fc fragment of IgG1, IgG3, or IgG4. The affinity of aglycosylated receptor for the IgG‐Fc fragment decreases only very moderately (1.4‐ to 1.9‐fold) compared to that of the glycosylated receptor as determined by surface plasmon resonance . As indicated above, FcγRI is the only FcγR displaying a third domain ( Fig.…”

Section: High Resolution Structure Of the High‐affinity Receptor Fcγrimentioning

confidence: 73%

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Structural analysis of Fc/FcγR complexes: a blueprint for antibody design

Caaveiro

Kiyoshi

Tsumoto

2015

Immunological Reviews

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The number of studies and the quality of the structural data of Fcγ receptors (FcγRs) has rapidly increased in the last few years. Upon critical examination of the literature, we have extracted general conclusions that could explain differences in affinity and selectivity of FcγRs for immunoglobulin G (IgG) based on structural considerations. FcγRs employ a little conserved asymmetric surface of domain D2 composed of two distinct subsites to recognize the well-conserved lower hinge region of IgG1-Fc. The extent of the contact interface with the antibody in subsite 1 of the receptor (but not in subsite 2), the geometrical complementarity between antibody and receptor, and the number of polar interactions contribute decisively toward strengthening the binding affinity of the antibody for the receptor. In addition, the uncertain role of the N-linked glycan of IgG for the binding and effector responses elicited by FcγRs is discussed. The available data suggest that not only the non-covalent interactions between IgG and FcγRs but also their dynamic features are essential for the immune response elicited through these receptors. We believe that the integration of structural, thermodynamic, and kinetic data will be critical for the design and validation of the next generation of therapeutic antibodies with enhanced effector capabilities.

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Section: High Resolution Structure Of the High‐affinity Receptor Fcγrimentioning

confidence: 73%

“…1). The relative affinity of FccRI for the Fc portion of IgG from different subclasses is given in Table 1 (16,26,54) and represented in Fig. 2.…”

Section: High Resolution Structure Of the High-affinity Receptor Fccrimentioning

confidence: 99%

Structural analysis of Fc/FcγR complexes: a blueprint for antibody design

Caaveiro

Kiyoshi

Tsumoto

2015

Immunological Reviews

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“…These findings were similar to the IgG–FcγRIa interaction results that were reported previously in the literature. 2 , 10 , 47 , 48 Protein A sensorgrams were not globally analyzed with a 1:1 interaction due to the presence of five potential target-binding domains. The steady-state K D values for protein A were in the range of 10.67–35.28 nM.…”

Section: Resultsmentioning

confidence: 99%

Characterization of FcγRIa (CD64) as a Ligand Molecule for Site-Specific IgG1 Capture: A Side-By-Side Comparison with Protein A

et al. 2022

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Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin–biotin interaction, and His-tagged FcγRIa capture. The His-tagged FcγRIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate FcγRIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the FcγRIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI revealed that the association rate (k a 50–80 × 105 M–1 s–1) increased in comparison to His capture method (1.9–2.4 × 105 M–1 s–1). In addition, the dissociation rate (k d 10–5 s–1) seemed slower over the His capture method (10–4 s–1) and provided stability on the chip surface during the dissociation phase. The K D values for FcγRIa were found in the picomolar range (2.1–10.33 pM from steady-state affinity analysis and 37.5–46.2 pM from kinetic analysis) for IgG1-type antibodies. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.

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“…was constructed from the pET-p7-MFcR vector to keep 326 the translation efficiency low[9]). Expression of soluble wild-type 327 rhEPOR (designated as EPORwt) was approximately 2 mg/l of cul-328 ture medium (optical density at 600 nm of culture: 5.37).329 …”

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confidence: 99%

“…To 70 develop an affinity ligand from rhEPOR, we applied the protein 71 engineering technique described in our previous report to the engi-72 neering of rhEPOR. 73 Materials and methods 74 Construction of vectors 75 To construct the pET-MalE21 vector, PCR was performed using 76 PrimeSTAR HS DNA Polymerase (Takara Bio, Shiga, Japan) and the 77 pET-p7-MFcR vector [9] as a template and primers 1 and 2 78 (Supplementary Table S1). This PCR product digested by XbaI and 79 NcoI was inserted into the XbaI-NcoI site of the pET-26b vector The EPORwt gene (encoding the extracellular domain of EPOR) 82 (DDBJ accession number: AB937115) was constructed by PCR using 83 the primers listed in Supplementary Table S1.…”

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confidence: 99%