TY - JOUR
T1 - Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides
AU - Sakamoto, Seiichi
AU - Pongkitwitoon, Benyakan
AU - Nakamura, Seiko
AU - Maenaka, Katsumi
AU - Tanaka, Hiroyuki
AU - Morimoto, Satoshi
N1 - Funding Information:
Japan Society for the Promotion of Science for Young Scientists, partial; Grant in Aid from the Japan Society for the Promotion of Science Asian CORE Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
PY - 2010/9
Y1 - 2010/9
N2 - A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 μg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli.
AB - A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 μg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli.
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U2 - 10.1093/jb/mvq072
DO - 10.1093/jb/mvq072
M3 - Article
C2 - 20592135
AN - SCOPUS:77956428396
SN - 0021-924X
VL - 148
SP - 335
EP - 340
JO - Journal of Biochemistry
JF - Journal of Biochemistry
IS - 3
ER -