Sagami Chemical Research Institute, Ayase, Kanagawa, Japan.
Appl Microbiol Biotechnol. 2012 May;94(4):1051-9. doi: 10.1007/s00253-012-3902-x.
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.
人 FcγRI（CD64）是一种整合膜糖蛋白，作为与单体 IgG 结合的高亲和力受体发挥作用。在这项研究中，FcγRI 的细胞外区域，即与 IgG 相互作用的实际部分，在大肠杆菌中作为糖基化重组人 FcγRI（rhFcγRI）表达。糖基化 rhFcγRI 的可溶性形式在大肠杆菌的周质中表达。通过低诱导水平增加可溶性糖基化 rhFcγRI 的产生。此外，通过修饰 rhFcγRI 的核糖体结合位点和起始密码子之间的假定区域来增加这种产生，融合表达载体的信号肽（MalE、PelB 或 TorT）。通过诱导和翻译效率的优化，可溶性糖基化 rhFcγRI 的产量高达约 0.8 mg/l 培养基。表面等离子体共振分析表明，糖基化 rhFcγRI 与人 IgG1（平衡解离常数 K D=[1.7±0.2]×10−10 M）和 IgG3（K D=[1.1±0.2]×10−10 M）的结合亲和力与糖基化 rhFcγRI 相似。
