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 6.6 L bioreactor. Briefly, a pH-stat feeding strategy was employed, and two different cell densities (OD600 of 46 and 100) were examined for the induction of FcγRIIa gene expression. When cells were induced at a high cell density (OD600 of 100), the cell density increased to an OD600 of 234 within 9 h after induction, and a 2-fold higher production yield was obtained compared with that of induction at low cell density (OD600 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.
Bibliographical noteFunding Information:
We thank Dr. George Georgiou (Univ. of Texas at Austin) for providing the plasmid. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant No. 2010-0011216 ). N. Velmurugan was supported by the BK21 Post-Doctoral Research Fund .
- Escherichia coli
- High cell density culture