title: Enhanced production of human FcγRIIa receptor by high cell density cultivation of Escherichia coli.
abstract: Human Fc receptors (FcγR) are membrane glycoproteins that are expressed on all immunologically active cells and have a well-defined role in regulating innate and adaptive immune responses by binding to the immunoglobulin G (IgG) antibody. Among the several classes of Fc receptors, FcγRIIa is the most widely expressed, and it serves as an important reagent in antibody engineering. Here, we report on high cell density cultivations (HCDC) of Escherichia coli for preparative scale production of FcγRIIa in a 6.6L bioreactor. Briefly, a pH-stat feeding strategy was employed, and two different cell densities (OD(600) of 46 and 100) were examined for the induction of FcγRIIa gene expression. When cells were induced at a high cell density (OD(600) of 100), the cell density increased to an OD(600) of 234 within 9h after induction, and a 2-fold higher production yield was obtained compared with that of induction at low cell density (OD(600) of 46). After simple purification steps including denaturation and refolding, 87.7 mg of soluble FcγRIIa that was more than 95% pure was obtained from a 20-mL culture with high recovery yield (≈54%). The biological activity of purified FcγRIIa was also confirmed by evaluating its interaction with all subclasses of IgG antibodies using an ELISA bioassay.
authors.affiliation: Department of Chemical and Biomolecular Engineering, KAIST, Yuseong-gu, Daejeon, Republic of Korea.
authors.forename: Natarajan
authors.initials: N
authors.lastname: Velmurugan
authors.forename: Hee Sung
authors.initials: HS
authors.lastname: Kim
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
mesh_terms: D019149:Bioreactors; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D017452:Receptors, IgG / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C080568:Fc gamma receptor IIA; D017452:Receptors, IgG; D011994:Recombinant Proteins