Novel strategy for expression and characterization of rabies virus glycoprotein

• Published in: Protein expression and purification Vol. 168; p. 105567
• Main Authors: Zhao, Rongqing, Shan, Yi, Li, Maohua, Lou, Zhiyong, Feng, Ye, Huang, Lisong, Ren, Wenlin, Wang, Panpan, Sun, Yufei, Sun, Ying, Su, Junchi, Sun, Hunter, Hong, Dee, Li, Yuhua, Chen, Ruifeng, Sun, Le
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2020
• Subjects: Animals; Antibodies, Neutralizing - biosynthesis; Antibodies, Viral - biosynthesis; Antigens, Viral - administration & dosage; Antigens, Viral - genetics; Antigens, Viral - immunology; Characterization; Cloning, Molecular; Cross Protection; ELISA; Escherichia coli - genetics; Escherichia coli - metabolism; Expression; Gene Expression; Genetic Vectors - chemistry; Genetic Vectors - metabolism; Glycoprotein; Humans; Immune Sera - chemistry; Immunoglobulin G - genetics; Immunoglobulin G - metabolism; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - metabolism; Mice; Protein Engineering - methods; Protein Sorting Signals - genetics; Rabies - prevention & control; Rabies - virology; Rabies Vaccines - administration & dosage; Rabies Vaccines - biosynthesis; Rabies Vaccines - genetics; Rabies virus; Rabies virus - genetics; Rabies virus - immunology; Recombinant Fusion Proteins - administration & dosage; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Viral Envelope Proteins - administration & dosage; Viral Envelope Proteins - genetics; Viral Envelope Proteins - immunology; Characterization; Expression; Rabies virus; Glycoprotein; ELISA
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2019.105567

Rabies is a fatal zoonosis which could affect all mammals. Glycoprotein (G protein) from the rabies virus plays an important role in the binding of virus to target cells. However, expression of the G protein with native conformation has been a great challenge for many years. In this study, we solved this problem by replacing the original signal peptide of rabies virus G protein with the one from the heavy chain of human IgG. The expression levels of recombinant G protein dramatically increased from a few μg/L to 50 mg/L in the culture supernatants. The identity of the recombinant G protein was confirmed by western blotting using both 6XHis mAb 6E2 and rabies G protein mAb 7G3. The correct conformation of the recombinant G protein was shown by using rabies virus neutralizing antibodies. In addition, the recombinant G protein had immune-reactivities with mice sera raised against rabies vaccines and vice versa. Taken together, our data suggested that by replacing the signal peptide, the expression level of the G protein with native conformation could be significantly improved. This would help the development of a rabies subunit vaccine, structural studies of rabies G protein, elucidation of the signal pathway of RABV infection. •A new method for improved expression of viral glycoproteins in mammalian cell system.•A stable CHO clone with several thousand folds higher RABV G protein expression at 50 mg/L.•The recombinant RABV G protein displayed ideal level of immunoreactivity and immunogenicity.•The recombinant RABV G protein is suitable for serology, vaccinology and structural studies.