Cloning and functional expression of a chitinase cDNA from the common cutworm, Spodoptera litura, using a recombinant baculovirus lacking the virus-encoded chitinase gene.
A Chitinase cDNA named Slchi was cloned from the epidermis of the common cutworm, Spodoptera litura, and the enzymatic properties of its recombinant proteins were characterized. The Slchi cDNA encodes 552 amino-acid residues (aa) including a 19 aa putative signal peptide, with the calculated molecular mass of the putative mature protein 60,152 Da. A major transcript of Slchi about 2.8 kb was detected in the epidermis only during molting in the last instar larvae, suggesting its involvement in the digestive system for old cuticle. The E. coli-produced recombinant Slchi exhibited weak chitinolytic activity against 4MU-(GlcNAc)(3)>4MU-(GlcNAc)(2)>4MU-(GlcNAc)(4), in this order, but not against 4MU-(GlcNAc)(1). A recombinant Slchi with higher specific activity was obtained using recombinant Hyphantria cunea NPV (HycuNPV), which expresses Slchi under polyhedrin promoter. To discriminate chitinase activity of recombinant Slchi from an active chitinase encoded in HycuNPV genome (chiA), we further knocked out the chiA gene from the recombinant virus. The recombinant Slchi expressed in insect cell culture showed a similar substrate specificity against 4MU-(GlcNAc)(n) (n=1-4) to that produced in E. coli, while the viral chitinase showed the highest activity against 4MU-(GlcNAc)(2). The recombinant Slchi was secreted rapidly into the culture medium from the infected cells, whereas the viral chitinase retained predominantly in the cells.
Shinoda
T
T
National Research Institute of Vegetables, Ornamental Plants and Tea, Ano, Mie 514-2392, Japan. shinoda@nivot.affrc.go.jp
Kobayashi
J
J
Matsui
M
M
Chinzei
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate; D018411:Spodoptera / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate; D006639:Histidine; D002688:Chitinases; C094590:chitinase ChiA