Department of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan.
J Biochem. 2021 Oct 11;170(2):289-297. doi: 10.1093/jb/mvab039.
Pichia pastoris is a popular eukaryotic system employed for the fast, simple and inexpensive production of recombinant protein including biotherapeutics such as human albumin. The CH2 domain of human Immunoglobulin G (IgG) is a promising scaffold for developing novel therapeutics. To accelerate the research of CH2 domain, we have established a procedure to highly express human CH2 domain (∼150 mg/l) as well as human Fc (∼30 mg/l) in yeast P. pastoris. The procedure yields, simultaneously, a major glycosylated (∼70%) and non-glycosylated (∼30%) fractions. They can be easily separated with high purity. Although both forms of CH2 domain have essentially the same secondary structure, the presence of the glycan increased the thermal stability of the CH2 domain by about 5°C as determined from calorimetry. The purified glycosylated CH2 domain elicited polyclonal antibodies in mouse, recognizing not only the CH2 domain, but also recombinant human Fc and the commercial IgG1 antibody Rituxan. Protein A and Protein G binding to the kink region between CH2 domain and CH3 domain of human Fc are used to purify therapeutic proteins. Therefore, these antibodies are candidates to develop a novel affinity material to purify human antibodies using their CH2 domain.
毕赤酵母是一种常用的真核表达系统，用于快速、简单且廉价地生产重组蛋白，包括人血清白蛋白等生物疗法。人免疫球蛋白 G（IgG）的 CH2 结构域是开发新型治疗药物的有前途的支架。为了加速 CH2 结构域的研究，我们建立了一种在酵母毕赤酵母中高效表达人 CH2 结构域（约 150mg/L）和人 Fc（约 30mg/L）的方法。该方法同时产生主要糖基化（约 70%）和非糖基化（约 30%）两种形式。它们可以很容易地以高纯度分离。尽管两种形式的 CH2 结构域具有基本相同的二级结构，但糖基化的存在使 CH2 结构域的热稳定性提高了约 5°C，这是通过量热法确定的。纯化的糖基化 CH2 结构域在小鼠中引发了多克隆抗体，不仅能识别 CH2 结构域，还能识别重组人 Fc 和商业 IgG1 抗体利妥昔单抗。蛋白 A 和蛋白 G 与人 Fc 的 CH2 结构域和 CH3 结构域之间的转角区域结合，用于纯化治疗性蛋白。因此，这些抗体是开发一种新型亲和材料的候选物，可以利用其 CH2 结构域来纯化人抗体。
