Abstract
Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.
Highlights
Baculoviruses are known to infect over 660 insect host species
Cells lyse and die quickly in response to viral infection, and the cell secretory pathway is impaired during the late stage of viral infection, and recombinant protein production is typically restricted to three days [5]
We expect that establishing a new combination of baculovirus/insect cell expression systems may break through the limitations of the current BEVSs
Summary
Baculoviruses are known to infect over 660 insect host species. The availability of lepidopteran cell lines has steadily increased at about 50 new lines per decade, and more than 30 baculoviruses that have been grown in insect cell cultures have been reported [1]. Cells lyse and die quickly in response to viral infection, and the cell secretory pathway is impaired during the late stage of viral infection, and recombinant protein production is typically restricted to three days [5]. We doubt these limitations are universal in all kinds of baculovirus/insect cell interactions. An alphabaculovirus (lepidopteran-specific nucleopolyhedrovirus (NPV), such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has about 150 genes but only shares 38 core genes with other alphabaculoviruses [6] This means that over 100 genes in an alphabaculovirus are specific and may uniquely interact with their native host insect cells. We expect that establishing a new combination of baculovirus/insect cell expression systems may break through the limitations of the current BEVSs
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