The nucleoprotein of Tomato spotted wilt virus as protein tag for easy purification and enhanced production of recombinant proteins in plants.
Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These aggregates result from the homopolymerization of the N protein, and are highly stable in plant cells. These properties feature the N protein as a potentially useful protein fusion partner. To evaluate this potential, the N protein was fused to the Aequorea victoria green fluorescent protein (GFP), either at the amino or carboxy terminus, and expressed in plants from binary vectors in Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens and evaluated after 4 days, revealing an intense GFP fluorescence under UV light. Microscopic analysis revealed that upon expression of the GFP:N fusion a small number of large aggregates were formed, whereas N:GFP expression led to a large number of smaller aggregates scattered throughout the cytoplasm. A simple purification method was tested, based on centrifugation and filtration, yielding a gross extract that contained large amounts of N:GFP aggregates, as confirmed by GFP fluorescence and Western blot analysis. These results show that the homopolymerization properties of the N protein can be used as a fast and simple way to purify large amounts of proteins from plants.
Lacorte
Cristiano
C
Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.
Ribeiro
Simone G
SG
Lohuis
Dick
D
Goldbach
Rob
R
Prins
Marcel
M
D015153:Blotting, Western; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D009698:Nucleoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D017865:Tospovirus / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D009698:Nucleoproteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins