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.

Automated summary

Chimeric fusion proteins consisting of VHH and Fc fragments show increased accumulation when targeted to the chloroplast thylakoid lumen, indicating the potential of this compartment for stable folding of disulfide-containing recombinant proteins.