A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Processes

Chih-Yu Wu¹  Shih-Torng Ding¹  Chung-Hsiung Wang²  Chao-Wei Huang³  Yu-Shin Nai⁴  Pei-Yu Chu⁵ 

[1] Center of Biotechnology, National Taiwan University, Taipei 10617, Taiwan;Department and Graduate Institute of Entomology, National Taiwan University, Taipei 10617, Taiwan;Department of Animal Science and Technology, National Taiwan University, Taipei 10617, Taiwan;Department of Entomology, National Chung Hsing University, Taichung 402, Taiwan;Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 10617, Taiwan;

关键词: baculovirus expression system;    2A-mediated “cleavage” process;    recombinant porcine adiponectin;    multimer formation;    N-linked glycosylation;   

DOI  :  10.3390/pr7050291

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown