Molecular chaperone-assisted production of human alpha-1,4-N-acetylglucosaminyltransferase in silkworm larvae using recombinant BmNPV bacmids.
In this study, human alpha-1,4-N-acetylglucosaminyltransferase (alpha4GnT) fused with GFP(uv) (GFP(uv)-alpha4GnT) was expressed using both a transformed cell system and silkworm larvae. A Tn-pXgp-GFP(uv)-alpha4GnT cell line, isolated after expression vector transfection, produced 106 mU/ml of alpha4GnT activity in suspension culture. When Bombyx mori nucleopolyhedrovirus containing a GFP(uv)-alpha4GnT fusion gene (BmNPV-CP (-)/GFP(uv)-alpha4GnT) bacmid was injected into silkworm larvae, alpha4GnT activity in larval hemolymph was 352 mU/ml, which was 3.3-fold higher than that of the Tn-pXgp-GFP(uv)-alpha4GnT cell line. With human calnexin (CNX) or human immunoglobulin heavy chain-binding protein (BiP, GRP78) coexpressed under the control of the ie-2 promoter, alpha4GnT activity in larval hemolymph increased by 1.4-2.0-fold. Moreover, when BmNPV-CP (-)/GFP(uv)-alpha4GnT bacmid injection was delayed for 3 h after BmNPV-CP (-)/CNX injection, the alpha4GnT activity increased significantly to 922 mU/ml, which was 8.7-fold higher than that of the Tn-pXgp-GFP(uv)-alpha4GnT cell line. Molecular chaperone assisted-expression in silkworm larvae using the BmNPV bacmid is a promising tool for recombinant protein production. This system could lead to large-scale production of more complex recombinant proteins.
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
Makoto
M
Nakajima
Tatsuya
T
Kato
Shin
S
Kanamasa
Enoch Y
EY
Park
D000818:Animals; D012831:Bombyx / Q000502:physiology* / Q000821:virology; D000091342:Endoplasmic Reticulum Chaperone BiP; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000502:physiology*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases