Construction of a cysteine protease deficient Bombyx mori multiple nucleopolyhedrovirus bacmid and its application to improve expression of a fusion protein.
The bacmid system of BmMNPV with cysteine protease gene deletion (CPD-BmMNPV bacmid) was constructed using the lambda recombination system. The protease activities of Bombyx mori cells and silkworm larvae infected with this CPD-BmMNPV bacmid were reduced by 94% and 85%, respectively. By using this system, a GFP(uv)-beta1,3-N-acetylglucosaminyltransferase 2 (GFP(uv)-beta3GnT2) fusion protein was successfully expressed in silkworm larvae with less protein degradation and without larvae liquefaction; beta3GnT activity improved 30%. This CPD-BmMNPV bacmid system provides rapid protein production in silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation.
Hiyoshi
Masato
M
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
Kageshima
Ayano
A
Kato
Tatsuya
T
Park
Enoch Y
EY
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000821:virology; D002460:Cell Line; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D007814:Larva; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D017351:N-Acetylglucosaminyltransferases; D003546:Cysteine Endopeptidases