Purification and cDNA cloning of inducible antibacterial peptides from Protaetia brevitarsis (Coleoptera).
Three antibacterial peptides, named protaetins 1, 2, and 3, were purified and characterized from immunized larval hemolymph of Protaetia brevitarsis, a fruit tree pest in Korea. Also, protaetin 1 was cloned. Acid extraction, gel filtration, preparative acid-urea PAGE, and reversed-phase FPLC were used for purification of peptides. Protaetins 1 and 3 had molecular masses of 7.5 and 12 kDa on Tricine SDS-PAGE, respectively, and the molecular mass of protaetin 2 was 9,283.95 Da as determined by MALDI-TOF mass spectrometry. In an antibacterial assay, protaetins showed antibacterial activities against a panel of Gram-positive and -negative bacteria. For the RT-PCR (reverse transcription polymerase chain reaction) to obtain the complete primary sequence, the primer was designed according to the N-terminal amino acid sequence of protaetin 1. Amino acid sequence homology of protaetin 1 with holotricin 2, an antibacterial peptide from Holotrichia diomphalia, showed 99% identity. Northern blot analysis showed that the protaetin 1 gene was strongly expressed in the fat body after Escherichia coli injection, but not in normal fat body. Also, it was expressed in the gut, but was much weaker after immunization.
Yoon
Hye Suk
HS
Graduate School of Biotechnology, Korea University, Seoul, Korea.
Lee
Chang Seok
CS
Lee
Sang Yong
SY
Choi
Chung Sik
CS
Lee
In Hee
IH
Yeo
Sung Moon
SM
Kim
Hak R
HR
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D001517:Coleoptera / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006458:Hemolymph / Q000737:chemistry; D019476:Insect Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D007814:Larva / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010455:Peptides; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D019476:Insect Proteins; D010455:Peptides