Enhancement of correct protein folding in vivo by a non-lytic baculovirus.
The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP-LUC-ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.
Ho
Yu
Y
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan, Republic of China.
Lo
Huei-Ru
HR
Lee
Tzu-Ching
TC
Wu
Carol P Y
CP
Chao
Yu-Chan
YC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D007304:Insect Viruses / Q000235:genetics / Q000302:isolation & purification; D008156:Luciferases / Q000096:biosynthesis; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D012107:Research Design; D018411:Spodoptera / Q000166:cytology / Q000821:virology; D018406:Virus Physiological Phenomena; D014779:Virus Replication / Q000235:genetics
D008156:Luciferases