Expression of soluble and functional human neonatal Fc receptor in Pichia pastoris.
The neonatal Fc receptor (FcRn) is a non-covalently associated heterodimeric protein composed of a transmembrane anchored heavy chain (alphaFcRn) and a soluble light chain beta2-microglobulin (beta2m). In addition to its role in the transfer of maternal immunoglobulin Gs (IgGs) to the fetus, FcRn plays a key role in prolonging the serum half-life of IgGs in vivo. Herein, we report a strategy for functional expression of soluble human FcRn (shFcRn) in Pichia pastoris using a two-promoter vector system, where alphaFcRn and beta2m are co-expressed under their respective promoters in a single vector. The purified shFcRn from the culture supernatants correctly assembled to form the heterodimer with the typical secondary structures. At acidic pHs between 5.0 and 6.4, shFcRn exhibited substantial binding to the four subclasses of human IgGs at acidic pHs between 5.0 and 6.4, but at pHs between 6.8 and 8.0, its binding was negligible binding. No cross-reactivity with mouse IgG was exhibited even at acidic pH. This was consistent with the pH-dependent binding profiles of the shFcRn prepared from the mammalian cell expression. Furthermore, the shFcRn exhibited about 10-fold higher binding affinity with the tumor necrosis factor-alpha antagonists of monoclonal antibodies Infliximab and Adalimumab than that of Etanercept, providing a clue to their different serum half-lives in vivo. Our results suggest that the functionally expressed shFcRn from Pichia can be used for the biochemical and biological studies and as a screening probe for Fc engineering of human IgGs.
Lee
Chang-Han
CH
Dept of Molecular Science and Technology, Ajou University, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-749, Republic of Korea.
Choi
Dong-Ki
DK
Choi
Hye-Ji
HJ
Song
Moo-Young
MY
Kim
Yong-Sung
YS
D000068879:Adalimumab; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D061067:Antibodies, Monoclonal, Humanized; D000068800:Etanercept; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D000069285:Infliximab; D007700:Kinetics; D051379:Mice; D010843:Pichia / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011961:Receptors, Fc / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D012995:Solubility; D014170:Transformation, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D011961:Receptors, Fc; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D000069285:Infliximab; D000068879:Adalimumab; D000068800:Etanercept; C100688:Fc receptor, neonatal