Cloning and expression of the Momordica charantia trypsin inhibitor II gene in silkworm by using a baculovirus vector.
MCTI-II (Momordica charantia trypsin inhibitor II) isolated from bitter gourd (Momordica charantia LINN.) seeds is one of the serine protease inhibitors of the squash family. We cloned cDNA that encodes MCTI-II and constructed an expression system for MCTI-II by using a baculovirus vector. The recombinant baculovirus was inoculated to early fifth-instar larvae of the silkworm (strain: Shunrei x Shougetsu). Four days after infection, the hemolymph of silkworm larvae was collected and the recombinant protein was purified. Two kinds of expressed MCTI-II protein were obtained. An amino acid sequence analysis of the two proteins indicates that both were similar to the authentic inhibitor, except for the addition of a tripeptide derived from the vector at the N-terminus. One of the two inhibitors (MCTI-II A) resulted in a single PTH-amino acid in each Edman degradation cycle, while the other (MCTI-II B) resulted in two PTH-amino acids, suggesting the occurrence of cleavage of the reactive site. The inhibitory activities of MCTI-II expressed toward trypsin are examined in terms of the Ki value, these being 6.4 x 10(-10)M for MCTI-II A and 5.2 x 10(-10) M for MCTI-II B.
Sato
S
S
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Japan.
Kamei
K
K
Taniguchi
M
M
Sato
H
H
Takano
R
R
Mori
H
H
Ichida
M
M
Hara
S
S
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera; D014361:Trypsin Inhibitors / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D018076:DNA, Complementary; C093458:Momordica charantia trypsin inhibitor II'; D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors