Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli.
Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.
Cao
Wei
W
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China.
Zhou
Yuxun
Y
Ma
Yushu
Y
Luo
Qingping
Q
Wei
Dongzhi
D
D000577:Amides; D029845:Amphibian Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D000818:Animals; D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005658:Fungi / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects
D000577:Amides; D029845:Amphibian Proteins; D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; C485283:dermaseptin B2, Phyllomedusa bicolor