Virus-like particles (VLPs) and capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of capsomeres, and VLP assembly. In this study, three module designs of a rotavirus hydrophobic peptide (RV10) were synthesized using synthetic biology. Among the three synthetic modules, modularization of the murine polyomavirus VP1 with a single copy of RV10 flanked by long linkers and charged residues resulted in the expression of stable modular capsomeres. Further employing the approach of module titration of RV10 modules on each capsomere via Escherichia coli coexpression of unmodified VP1 and modular VP1-RV10 successfully translated purified modular capomeres into modular VLPs when assembled in vitro. Our results demonstrate that tailoring the physicochemical properties of modules to enhance modular capsomeres stability is achievable through synthetic biology designs. Combined with module titration strategy to avoid steric hindrance to intercapsomere interactions, this allows bioprocessing of bacterially produced in vitro assembled modular VLPs.
Three lipases from different sources (Pseudomonas aeruginosa MTCC 5113 (PAL), porcine pancreatic (PPL) and Candida rugosa (CRL)) were screened for the transesterification of DL-3-phenyllactic acid (DL-3-PLA) in three different ionic liquids (1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIM][BF 4 ]), 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIM][PF 6 ]) and 1-ethyl-3-methyl imidazolium tetrafluoroborate ([EMIM][BF 4 ])) in combination with different organic solvents (e.g. hexane, cyclohexane, isooctane, toluene, vinyl acetate and dichloromethane). CRL efficiently catalyzed the esterification of DL-3-PLA to L-O-acetyl-3-PLA using vinyl acetate as acyl donor, showing excellent enantioselectivity (EÂ100) and conversion in all three ionic liquids. PPL exhibited enantioselectivity only in the presence of [BMIM][BF 4 ] and was not able to catalyze DL-3-PLA esterification in the other two ionic liquids. PAL did not show any reactivity with DL-3-PLA in any of the ionic liquids. Various parameters such as solvent and ionic liquid concentration, reaction time, substrate and enzyme concentration on the activity and enantioselectivity of CRL were studied.
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