Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus-like particle

Abstract

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 co-expression 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.

Keywords: Escherichia coli; co-expression; linkers; module titration; rotavirus; synthetic biology.

Figure 1

Construct designs for modular capsomeres. (A) VLP platform with engineered insertion site at VP1 surface‐exposed S4 loop,8 with VP1 protein expressed as GST fusion protein. (B) Modular constructs VLP‐(RV10)₃, VLP‐(RV10)₃ESE, and VLP‐RV10 with inserted modules (RV10)₃, (RV10)₃ESE, and G4S‐Q25‐E4‐RV10‐E4‐P6‐G4S at S4 loop of VP1, respectively. (C) Capsomere platform with engineered N‐terminus, S1 loop, S4 loop and C‐terminus insertion sites on truncated VP1,10 expressed as GST fusion protein. (D) Modular constructs Cap(RV10)₃ and Cap(RV10)₃ESE with inserted modules (RV10)₃ and (RV10)₃ESE, respectively, at N‐terminus, S4 loop and C‐terminus insertion sites of truncated VP1. (E) Dual expression construct, pET‐VP1‐RV10, carrying both wild‐type VP1 and modular VP1‐G4S‐Q25‐E4‐RV10‐E4‐P6‐G4S sequence.

Figure 2

Expression and protein solubility of GST fusion target proteins. The target proteins were detected and visualized by SDS‐PAGE from un‐induced cultures (U), total cell lysate (T), and soluble fraction (S) of induced cultures. Novex sharp pre‐stained protein marker (M) was used as a ladder. Arrows indicate the GST‐tagged target proteins.

Figure 3

Downstream processing of capsomeres. (A) Size exclusion chromatograms of modular capsomeres following TEVp‐mediated release of GST tag. P1, P2, and P3 represent the aggregate, capsomere, and GST peaks, respectively. (B) SDS‐PAGE analysis on downstream processing of capsomeres. Marker protein (M), GST‐tagged soluble protein after GST affinity purification (S), TEVp‐mediated tag cleavage (D), P1 aggregate peak fraction (A), and P2 capsomere peak fraction (C).

Figure 4

Characterization of capsomeres and in vitro assembled products. (A) SEC‐HPLC/LS analysis of VP1 and VLP‐RV10 capsomeres following TEVp‐mediated release of GST tag. P1, P2, and P3 represent peaks for VP1 capsomeres, VLP‐RV10 capsomeres, and GST dimers, respectively. (B) AF4 fractograms of in vitro assembled products of VP1, VLP‐(RV10)₃ESE, and VLP‐RV10. P1, P2, and P3 represent peaks for non‐assembled proteins, assembled VLPs and aggregates, respectively.

Figure 5

Co‐expression strategy for bacterially produced stable modular capsomeres and in vitro assembled modular VLPs presenting module RV10. (A) Detection and visualization of non‐tagged target proteins of pET‐VP1 and pET‐VP1‐RV10 from cell lysates. (B) Size exclusion chromatograms of pET‐VP1 and pET‐VP1‐RV10 capsomeres following purification by selective salting‐out precipitation. P1, P2, and P3 represent the aggregate, capsomere, and co‐precipitated E. coli protein peaks, respectively. (C) SDS‐PAGE analysis of the target proteins. Marker protein (M), purified protein by selective salting‐out precipitation (P), P1 aggregate peak fraction (A), and P2 capsomere peak fraction (C). (D) AF4 fractograms and TEM micrographs of assembled products (i) VP1VLP and (ii) RV10VLP. P1, P2, and P3 represent peaks for non‐assembled proteins, assembled VLPs, and aggregates, respectively.