scholarly journals FlexiBAC: a versatile, open-source baculovirus vector system for protein expression, secretion, and proteolytic processing

2018 ◽  
Author(s):  
Régis P. Lemaitre ◽  
Aliona Bogdanova ◽  
Barbara Borgonovo ◽  
Jeffrey B. Woodruff ◽  
David N. Drechsel

ABSTRACTBaculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-β family growth factors. To overcome these limitations, we engineered “FlexiBAC”, a system that simplifies baculovirus production and permits furin-driven proteolytic maturation of targets. This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 146 shuttle vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. We demonstrate that this system can be used to produce high levels of mature, active forms of TGF-β family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains.

Processes ◽  
2019 ◽  
Vol 7 (5) ◽  
pp. 291 ◽  
Author(s):  
Chih-Yu Wu ◽  
Chao-Wei Huang ◽  
Yu-Shin Nai ◽  
Pei-Yu Chu ◽  
Chung-Hsiung Wang ◽  
...  

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.


Biochemistry ◽  
1990 ◽  
Vol 29 (9) ◽  
pp. 2295-2303 ◽  
Author(s):  
Ursula A. Germann ◽  
Mark C. Willingham ◽  
Ira Pastan ◽  
Michael M. Gottesman

2021 ◽  
Author(s):  
Adeline Harant ◽  
Hsuan Pai ◽  
Toshiyuki Sakai ◽  
Sophien Kamoun ◽  
Hiroaki Adachi

Abstract Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis thaliana. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of nucleotide-binding leucine-rich repeat (NLR) plant immune receptors has greatly advanced following the discovery of the Arabidopsis HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome. Here, we describe a vector system of 72 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs (CC-NLRs) for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. Effector-activated and autoactive NbZAR1 trigger the cell death response in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana, thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source pZA plasmid collection forms an additional experimental system to Arabidopsis for in planta resistosome studies.


2000 ◽  
Vol 20 (2) ◽  
pp. 265-273 ◽  
Author(s):  
Tapas Das ◽  
Paul W. Johns ◽  
Vincent Goffin ◽  
Paul Kelly ◽  
Bruce Kelder ◽  
...  

1994 ◽  
Vol 136 (3-4) ◽  
pp. 375-380 ◽  
Author(s):  
S. J. Edwards ◽  
M. B. Hayden ◽  
R. C. Hamilton ◽  
J. A. Haynes ◽  
I. T. Nisbet ◽  
...  

Sign in / Sign up

Export Citation Format

Share Document