Efficient expression of recombinant soluble human FcγRI in mammalian cells and its characterization.
The extracellular domain of human FcγRI which interacts with a human IgG was expressed as recombinant soluble human FcγRI (rshFcγRI) by Chinese hamster ovary (CHO) cell. Stable CHO cell clones with efficient expression of rshFcγRI were established based on a dihydrofolate reductase (DHFR)/methotrexate (MTX) gene-amplification system. The CHO clones efficiently produced rshFcγRI under high-density continuous culture in a bioreactor. After 53 days of culture, the number of cells had reached approximately 4 × 10⁶ cells/mL in the bioreactor and the average production of rshFcγRI had reached 7.4 mg L-medium⁻¹ day⁻¹. Secreted rshFcγRI was purified to a homogeneous state using cation exchange and affinity chromatographies. The binding affinities of rshFcγRI to human IgG subclasses were determined using surface plasmon resonance analysis. The binding affinities of rshFcγRI to human IgG1/κ and IgG3/κ were high (1.59 × 10⁻¹⁰ and 2.81 × 10⁻¹⁰ M, respectively), whereas that of rshFcγRI to human IgG4/κ was lower binding affinity (1.41 × 10⁻⁸ M). Binding to IgG2/κ was not detectable. Examination of circular dichroism spectra indicated that rshFcγRI was rich in β-structures and loop or turn structures, but there were few α-helices. These results may be valuable for further studies of the structure and function of human FcγRI.
Tosoh Corporation, 2743-1 Hayakawa, Ayase-shi, Kanagawa 252-1123, Japan. yoshiharu-asaoka-zy@tosoh.co.jp
Yoshiharu
Y
Asaoka
Toru
T
Tanaka
Kanta
K
Tsumoto
Masahiro
M
Tomita
Teruhiko
T
Ide
D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000295:instrumentation; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D004867:Equipment Design; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D017452:Receptors, IgG / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility
D007074:Immunoglobulin G; D017452:Receptors, IgG; D011994:Recombinant Proteins