Molecular cloning, expression in Escherichia coli of Attacin A gene from Drosophila and detection of biological activity

• Published in: Molecular biology reports 2010-06, Vol.37 (5), p.2463-2469
• Main Authors: Wang, Li-Na, Yu, Bing, Han, Guo-Quan, Chen, Dai-Wen
• Format: Article
• Language: English
• Subjects: Animals; Antibacterial activity; Antimicrobial activity; antimicrobial peptides; Attacin A; Cloning; Cloning, Molecular; Drosophila; Drosophila melanogaster - drug effects; Drosophila melanogaster - genetics; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Drosophila Proteins - pharmacology; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - metabolism; Expression vectors; Fusion protein; Gel electrophoresis; Genes, Insect - genetics; Genetic Vectors; Genetics; Hemolysis; Hemolysis activity; Immunity; Insects; Microbial Sensitivity Tests; Molecular biology; Molecular Sequence Data; Open reading frames; Peptides; Peptides - pharmacology; Plasmids; Polymerase chain reaction; Prokaryotic Cells - drug effects; Prokaryotic Cells - metabolism; Prokaryotic expression; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Restriction Mapping; RNA; Sequence Analysis, DNA
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/s11033-009-9758-1

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS-PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS-PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.