In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system

Abstract

Baculovirus expression vector system (BEVS) has been recognized as a potent protein expression system in engineering valuable enzymes and vaccines. Various fusion tags facilitate protein purification, leaving the potential risk to influence the target protein’s biological activity negatively. It is of great interest to consider removing the additional tags using site-specific proteases, such as human rhinoviruses (HRV) 3C protease. The current study validated the cleavage activity of 3C protease in Escherichia coli and silkworm-BEVS systems by mixing the cell or fat body lysates of 3C protein and 3C site containing target protein in vitro. Further verification has been performed in the fat body lysate from co-expression of both constructs, showing remarkable cleavage efficiency in vivo silkworm larvae. We also achieved the glutathione-S-transferase (GST) tag-cleaved product of the VP15 protein from the White spot syndrome virus after purification, suggesting that we successfully established a coinfection-based recognition-and-reaction BEVS platform for the tag-free protein engineering.

Display of multiple proteins on engineered canine parvovirus-like particles expressed in cultured silkworm cells and silkworm larvae

“…The VP2-derived VLPs were obtained in mg scale per 10 silkworm larvae and self-assembled to an intact structural formation, ensuring convincible VLP display results in vitro and in vivo via co-infection with the recombinant baculovirus mixture, as demonstrated in Figure 2D ; Figures 4B, C . This approach is remarkably efficient for producing protein complexes and timesaving for verifying in vivo enzymatic reactions like specific protease ( Xu et al, 2022 ) and the covalent protein conjugation in this study ( Xu et al, 2019 ). It still requires further consideration to apply coinfection or in vitro assembly in a case-dependent manner since there is a potential risk for the coupled VP2-antigen protein complex not to be self-assembled into VLP, which is also true for validating the status of each antigen display on VLP surfaces.…”

“…Expression and purification of all proteins in cultured Bm5 cells or silkworm larvae were performed according to our previous reports ( Xu et al, 2019 ; Xu et al, 2022 ). Briefly, the fat body from silkworm larvae at 5 days post-infection (dpi) was resuspended and sonicated in a lysis buffer (100 mM Tris-HCl pH 8.4, 0.15 M NaCl, 1 mM EDTA, 0.1% NP-40, 1 × proteinase inhibitor (Roche, Tokyo, Japan)).…”

“…In scale-up of production, it is convenient to use silkworm-BEVS since a stable and cost-effective rearing method for domestic has already been developed in many Asian contourites ( Kato et al, 2010 ). Moreover, it is rational to consider that each genetically homogenized silkworm can be considered an independent bioreactor, and individual differences in the productivity of recombinant POIs are less likely to occur ( Yamashita et al, 2017 ; Masuda et al, 2022 ; Xu et al, 2022 ). However, systematic manufacturing standards and regulations, including Good Manufacturing Practice (GMP), are still under construction for the silkworm-BEVS system to meet the commercial level like well-developed protein production platforms, such as E. coli , yeast, and CHO cells ( Tomita, 2018 ).…”

Virus-like particles vaccines based on glycoprotein E0 and E2 of bovine viral diarrhea virus induce Humoral responses

“…Although mammalian cells produce fewer proteins, they have more complex and accurate PTM capabilities ( Zhu, 2012 ). BEVS has also become a mature production platform of VLPs vaccines and gene therapy vectors ( Felberbaum, 2015 ; Xu et al, 2022 ). Approved vaccines derived from baculovirus expression, including human use (Cervarix®, Glybera®, Flublok® and Provenge®) and veterinary use (BAYOVAC CSF E2®, Circumvent® PCV, Porcilis® Pesti, Porcilis® PCV and Ingelvac CircoFLEX®) ( Felberbaum, 2015 ; Xu et al, 2022 ).…”

“…BEVS has also become a mature production platform of VLPs vaccines and gene therapy vectors ( Felberbaum, 2015 ; Xu et al, 2022 ). Approved vaccines derived from baculovirus expression, including human use (Cervarix®, Glybera®, Flublok® and Provenge®) and veterinary use (BAYOVAC CSF E2®, Circumvent® PCV, Porcilis® Pesti, Porcilis® PCV and Ingelvac CircoFLEX®) ( Felberbaum, 2015 ; Xu et al, 2022 ). BEVS platform has many advantages, including manufacturing speed, flexible product design, inherent security and expansibility, which have attracted strong interest of researchers.…”