A VHH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7.

Chimeric fusion proteins comprising a single domain antibody (VHH) fused to a crystallizable fragment (Fc) of an immunoglobulin are modular glycoproteins that are becoming increasingly in demand because of their value as diagnostics, research reagents and passive immunization therapeutics. Because ER-associated degradation and misfolding may potentially be limiting factors in the oxidative folding of VHH-Fc fusion proteins in the ER, we sought to explore oxidative folding in an alternative sub-compartment, the chloroplast thylakoid lumen, and determine its viability in a molecular farming context. We developed a set of in-house expression vectors for transient transformation of Nicotiana benthamiana leaves that target a VHH-Fc to the thylakoid lumen via either secretory (Sec) or twin-arginine translocation (Tat) import pathways. Compared to stromal [6.63 ± 3.41 mg/kg fresh weight (FW)], cytoplasmic (undetectable) and Tat-import pathways (5.43 ± 2.41 mg/kg FW), the Sec-targeted VHH-Fc showed superior accumulation (30.56 ± 5.19 mg/kg FW), but was less than that of the ER (51.16 ± 9.11 mg/kg FW). Additionally, the introduction of a rationally designed de novo disulfide bond enhances in planta accumulation when introduced into the Sec-targeted Fc fusion protein from 50.24 ± 4.08 mg/kg FW to 110.90 ± 6.46 mg/kg FW. In vitro immunofluorescent labeling assays on VHH-Fc purified from Sec, Tat, and stromal pathways demonstrate that the antibody still retains VHH functionality in binding Escherichia coli O157:H7 and neutralizing its intimate adherence to human epithelial type 2 cells. These results overall provide a proof of concept that the oxidative folding environment of the thylakoid lumen may be a viable compartment for stably folding disulfide-containing recombinant VHH-Fc proteins. Copyright © 2021 Chin-Fatt and Menassa.

Citation

Adam Chin-Fatt, Rima Menassa. A VHH-Fc Fusion Targeted to the Chloroplast Thylakoid Lumen Assembles and Neutralizes Enterohemorrhagic E. coli O157:H7. Frontiers in plant science. 2021;12:686421

PMID: 34122494

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