Protein engineering of novel proteinase inhibitors and their effects on the growth of Spodoptera exigua larvae.
Novel types of proteinase inhibitors with multi-inhibitory activities were generated by replacement of phytocystatin domains in sunflower multi-cystatin (SMC) by the serine proteinase inhibitor BGIT from bitter gourd seeds. Two chimeric inhibitors SMC-T3 and SMC-T23, in which the third domain in SMC and the second and third domains in SMC were replaced by BGIT, acquired trypsin inhibitory activity (Ki: 1.46 x 10(-7) M and 1.75 x 10(-7) M), retaining inhibitory activity toward papain (Ki: 4.5 x 10(-8) M and 1.52 x 10(-7) M), respectively. We compared the chimeric inhibitors and the recombinant SMC (r-SMC) in relation to their effects on the growth of larval Spodoptera exigua. When the second instar larvae were reared on a diet containing rSMC, SMC-T3, or SMC-T23 for ten days, a significant reduction in weight gain was observed. Mean weights for rSMC, SMC-T3, and SMC-T23 were 43 mg, 32 mg, and 43 mg, respectively, as compared with that (60 mg) for the absence of the inhibitor. In contrast, BGIT had little effect on the growth of the S. exigua larvae. This result indicated that the chimeric inhibitor SMC-T3 with two phytocystatin domains and one serine proteinase inhibitor domain is an efficient inhibitor of proteinases in the S. exigua larvae. Therefore, this novel type of proteinase inhibitor with multi-inhibitory activities may represent a promising protein for successful application to a transgenic plant with insect resistance.
Inanaga
H
H
Laboratory of Biochemisty, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
Kobayasi
D
D
Kouzuma
Y
Y
Aoki-Yasunaga
C
C
Iiyama
K
K
Kimura
M
M
D000818:Animals; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D015891:Cystatins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D018744:DNA, Plant / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006368:Helianthus / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D018411:Spodoptera / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics
D015891:Cystatins; D018744:DNA, Plant; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors