Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli.
Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli. Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (muRPC PC 2.1/3). In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin. The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively. In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons. Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall. CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay. CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry.
Chae
K S
KS
Department of Biology, Korea University, Seoul, South Korea.
Lee
I H
IH
Choi
C S
CS
Kim
H R
HR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D002686:Chitin / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000187:drug effects; D006386:Hemagglutination Tests; D006458:Hemolymph / Q000737:chemistry*; D019476:Insect Proteins / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D016982:Micrococcus luteus / Q000187:drug effects; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000737:chemistry*; D011817:Rabbits; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012710:Serum Albumin, Bovine / Q000737:chemistry
D000596:Amino Acids; D002352:Carrier Proteins; D019476:Insect Proteins; D002686:Chitin; D012710:Serum Albumin, Bovine