Cargo-loading of hybrid cowpea chlorotic mottle virus capsids via a co-expression approach

Capsids of the cowpea chlorotic mottle virus (CCMV) are great candidates for the development into in vivo catalytic or therapeutic nanocarriers. However, due to their limited intrinsic stability at physiological pH, thus far no methods exist for incorporating cargo into these nanoparticles in cellulo. Here, we employ a stabilized VW1-VW8 ELP-CCMV variant for the development of a co-expression-based cargo-loading approach. Co-expression of the non-functionalized VW1-VW8 ELP-CCMV coat protein with fusion proteins with enhanced green fluorescent protein (mEGFP) and pyrrolysine synthase D (PylD) in E. coli enabled the purification of cargo-loaded capsids from the bacteria directly either via affinity chromatography or PEG-precipitation and subsequent size exclusion chromatography. Microscopy results indicated that the co-expression does not harm the E. coli cells and that proper folding of the mEGFP domain is not hampered by the co-assembly. Our co-expression strategy is thus a suitable approach to produce cargo-loaded CCMV nanoparticles.