Citation
- Authors: Li X. et al.
- Year: 2022
- Journal: J Am Chem Soc 145 322-333
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
The heavy chain and the light chain of the antibodies were respectively cloned into plasmid vector pcDNA3.1 between the cleavage sites 5′NheI and 3′NotI, both of which were then transfected into Expi 293F cells using FectoPro transfection kit (Polyplus transfection) and cultured in Expi293 Expression Medium for 5–7 days for expression. Next, the supernatants of cell cultures were harvested. The recombinant monoclonal antibodies were subsequently purified using a MabPurix Protein A column (Sepax) as instructed by the manufacturer, with a yield of 50–70 mg per liter.
Abstract
Alternative antibacterial therapies refractory to existing mechanisms of antibiotic resistance are urgently needed. One such attractive therapy is to inhibit bacterial adhesion and colonization. Ser O-heptosylation (Ser O-Hep) on autotransporters of Gram-negative bacteria is a novel glycosylation and has been proven to be essential for bacterial colonization. Herein, we chemically synthesized glycopeptides containing this atypical glycan structure and an absolute C6 configuration through the assembly of Ser O-Hep building blocks. Using glycopeptides as haptens, we generated first-in-class poly- and monoclonal antibodies, termed Anti-SerHep1a and Anti-SerHep1b, that stereoselectively recognize Ser O-heptosylation (d/l-glycero) with high specificity in vitro and in vivo. Importantly, these antibodies effectively blocked diffusely adhering Escherichia coli 2787 adhesion to HeLa cells and in mice in a dose- and Ser O-Hep-dependent manner. Together, these antibodies represent not only useful tools for the discovery of unknown serine O-heptosylated proteins bearing various C6 chiral centers but also a novel class of antiadhesion therapeutic agents for the treatment of bacterial infection.