A novel method for viral display of ER membrane proteins on budded baculovirus.
The baculovirus expression system has been used to express large quantities of various proteins, including membrane receptors. Here, we reveal a novel property of this expression system to be that certain membrane proteins can be displayed on the budded virus itself. We introduced the genes encoding sterol regulatory element-binding protein-2 (SREBP-2) or SREBP cleavage-activating protein (SCAP), important integral membrane proteins of the endoplasmic reticulum (ER) and/or the Golgi apparatus related to cellular cholesterol regulation, into a baculovirus vector. When insect cells were infected with SREBP-2 or SCAP recombinant viruses, it was found that these ER membrane proteins appeared on the budded baculovirus in addition to the host cell membrane fraction. Compared to proteins expressed on the cell membrane, membrane proteins displayed on virus exhibited both less aggregation and less degradation upon immunoblotting. Using this viral displayed SCAP as the screening antigen, we then generated a new monoclonal antibody specific against SCAP, which was useful for immunological localization studies. This system, which takes advantage of the viral display of membrane proteins, should prove to be a powerful additional tool for postgenomic protein analysis.
Urano
Yasuomi
Y
Department of Molecular Biology, Research Center for Advanced Science and Technology, The University of Tokyo, #35 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan.
Yamaguchi
Mineko
M
Fukuda
Rie
R
Masuda
Kazuyuki
K
Takahashi
Kazuaki
K
Uchiyama
Yasutoshi
Y
Iwanari
Hiroko
H
Jiang
Shu-Ying
SY
Naito
Makoto
M
Kodama
Tatsuhiko
T
Hamakubo
Takao
T
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics*; D018411:Spodoptera; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors / Q000235:genetics*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors