期刊
BIOSCIENCE REPORTS
卷 42, 期 6, 页码 -出版社
PORTLAND PRESS LTD
DOI: 10.1042/BSR20220739
关键词
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资金
- JSPS KAKENHI [20H00411]
The Baculovirus expression vector system (BEVS) is an effective protein expression system and has been used to engineer important enzymes and vaccines. The use of fusion tags for protein purification can negatively affect the biological activity of the target protein. This study validated the cleavage activity of the human rhinoviruses (HRV) 3C protease in both Escherichia coli and silkworm-BEVS systems. The findings demonstrate the successful establishment of a tag-free protein engineering platform using a coinfection-based recognition-and-reaction BEVS system.
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.
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