Human super antibody to viral RNA-dependent RNA polymerase produced by a modified Sortase self-cleave-bacteria surface display system
Issued Date
2023-12-01
Resource Type
eISSN
14752859
Scopus ID
2-s2.0-85179961676
Pubmed ID
38110987
Journal Title
Microbial Cell Factories
Volume
22
Issue
1
Rights Holder(s)
SCOPUS
Bibliographic Citation
Microbial Cell Factories Vol.22 No.1 (2023)
Suggested Citation
Glab-ampai K., Mahasongkram K., Chulanetra M., Saenlom T., Thueng-in K., Sookrung N., Chaicumpa W. Human super antibody to viral RNA-dependent RNA polymerase produced by a modified Sortase self-cleave-bacteria surface display system. Microbial Cell Factories Vol.22 No.1 (2023). doi:10.1186/s12934-023-02267-z Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/95835
Title
Human super antibody to viral RNA-dependent RNA polymerase produced by a modified Sortase self-cleave-bacteria surface display system
Author's Affiliation
Corresponding Author(s)
Other Contributor(s)
Abstract
Background: RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production. Methods and results: BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp′)-OmpA′, SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34. Conclusion: The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form.