High-level expression of human CH2 domain from the Fc region in Pichia pastoris and preparation of anti-CH2 antibodies

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 (similar to 150 mg/l) as well as human Fc (similar to 30 mg/l) in yeast P. pastoris. The procedure yields, simultaneously, a major glycosylated (similar to 70%) and non-glycosylated (similar to 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 degrees 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. [GRAPHICS] .

Automated summary

The study established a procedure to highly express human CH2 domain and human Fc in yeast P. pastoris, resulting in glycosylated and non-glycosylated forms that can be easily separated with high purity. It was found that glycosylation increased the thermal stability of the CH2 domain, and the purified glycosylated CH2 domain elicited polyclonal antibodies in mice. These antibodies could be potential candidates for developing a novel affinity material to purify human antibodies using their CH2 domain.