Improved expression of fusion protein using a cysteine- protease- and chitinase-deficient Bombyx mori (silkworm) multiple nucleopolyhedrovirus bacmid in silkworm larvae.
A bacmid system of BmMNPV [Bombyx mori (silkworm) multiple nucleopolyhedrovirus] with both the cysteine-protease and chitinase genes deleted (BmMNPV-CP(-)-Chi(-)) was constructed. The viral protease and chitinase activities in the haemolymph of Bombyx mori larvae infected with this BmMNPV-CP(-)-Chi(-) bacmid were reduced by 95 and 50% respectively. By using this system, a GFP(uv)-beta3GnT2 (green fluorescent protein excited by UV light-beta1,3-N-acetylglucosaminyltransferase 2) fusion protein was successfully expressed in silkworm larvae with less protein degradation and without larvae liquefaction; beta3GnT2 activity improved 2.8-fold over that of unmodified bacmid. This BmMNPV-CP(-)-Chi(-) bacmid system provides rapid protein production in silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation.
Park
Enoch Y
EY
Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan. yspark@agr.shizuoka.ac.jp
Abe
Takahiro
T
Kato
Tatsuya
T
D000818:Animals; D012831:Bombyx / Q000502:physiology*; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D007814:Larva / Q000235:genetics / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D002688:Chitinases; D010447:Peptide Hydrolases; D003545:Cysteine