Production of Hev b5 as a fluorescent biotin-binding tripartite fusion protein in insect cells.
The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.
Nordlund
Henri R
HR
Department of Biological and Environmental Science, NanoScience Center, P.O. Box 35, FIN-40014 University of Jyväskylä, Finland. henri.nordlund@uta.fi
Laitinen
Olli H
OH
Uotila
Sanna T H
ST
Kulmala
Minna
M
Kalkkinen
Nisse
N
Kulomaa
Markku S
MS
D000485:Allergens / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D052179:Antigens, Plant; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D002846:Chromatography, Affinity; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000302:isolation & purification; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D000485:Allergens; D052179:Antigens, Plant; D017931:DNA Primers; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C101800:allergen Hev b 5; D049452:Green Fluorescent Proteins; D001710:Biotin