Molecular cloning and characterization of cecropin from the housefly (Musca domestica), and its expression in Escherichia coli.
A cDNA encoding housefly (Musca domestica) cecropin transcript was isolated from total RNA using RT-PCR, 3'RACE, and lambda gt11 cDNA library screening, and was expressed in Escherichia coli. This is the first report of a cecropin nucleotide and amino acid sequence in the housefly. The open reading frame of Md-Cec (189bp) encodes a precursor of 63 aa, which is comprised of a 23 aa signal peptide and a 40 aa mature peptide. In terms of amino acid sequence, Md-Cec shares a high degree of identity (74-82%) with those of some Diptera insects. Northern blot, RT-PCR and in situ hybridization analyses revealed that the prececropin was temporally expressed 5h after bacteria-challenge in larvae, and was induced in the fat body, epithelia of the body wall, and the epidermis of the midgut. The DNA fragment encoding mature Md-Cec was then subcloned into the pGEX-4T-1 expression vector and was highly expressed in E. coli BL21 with IPTG induction. The expressed proteins, fused to glutathion S-transferase, were purified by glutathion-Sepharose 4B affinity chromatography and cleaved with thrombin, followed by gel filtration chromatography. Recombinant Md-Cec exhibited antimicrobial activity against E. coli.
Liang
Yongli
Y
School of Life Sciences, Shandong University, Jinan, Shandong 250100, People's Republic of China.
Wang
Jin-Xing
JX
Zhao
Xiao-fan
XF
Du
Xin-Jun
XJ
Xue
Jian-Feng
JF
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006793:Houseflies / Q000737:chemistry / Q000235:genetics*; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D019476:Insect Proteins; D012333:RNA, Messenger; C000595275:cecropin protein, Musca domestica