Recombinant Expression Of Antimicrobial Peptide CAD

Antimicrobial peptides (AMPs) are small molecular peptides which have antimicrobialactivity. AMPs were potential candidates of antibiotics without causing drug assistance.Cecropin AD (CAD), a cationic AMP, is composed of the first11residues of cecropin A andthe last26ones of cecropin D. CAD was found to have strong antimicrobial activity againstboth Gram-positive and Gram-negative bacteria, CAD also has the same effect as antibioticsin animal feeding tests. Several methods have been used to express CAD recently, but thesemethods are expensive due to the complicated purification process. This paper is calculated todevelop a costless and effective technology for commercial production of CAD.Firstly, the gene encoding CAD was synthesized based on the amino acid sequence. TheCAD gene was inserted into the genome of Kluyveromyces lactis GG799to express andsecrete CAD. But no antimicrobial activity was detected in the culture medium. Then wefused SUMO (Smt3) with CAD and used cationic elastin-like polypeptides (CELP) and Histag to purify fusion proteins respectively. We successfully constructed the recombinant strainsBL21/pET28a-CELP-SUMO-CAD and BL21/pET28a-6H-SUMO-CAD. The proteinCELP-SUMO-CAD was purified by three rounds of inverse transition cycling, and6H-SUMO-CAD was isolated by Ni-NTA. The two fusion proteins couldn’t be cleaved bySUMO protease Ulp1, CAD wasn’t released. It was hypothesized that the antimicrobialpeptide CAD influenced the conformation of SUMO, which made the Ulp1could notrecognize the conformation of SUMO. To remove the CELP tag, CAD was fused with CELPdirectly, and the enterokinase cleavage sequence was inserted between CAD and CELP. Therecombinant strain BL21/pET28a-CELP-ENK-CAD was constructed and the proteinCELP-ENK-CAD was isolated by inverse transition cycling. The protein CELP-ENK-CADwas cleaved by enterokinase and1.2mg high purity CAD (95%purity) were obtained from100mL culture medium after a hot spin and desalting. The results of bacteriostatic testsindicated the desalted CAD has the strong antibacterial activity against E.coli DH5α andS.aureus, The minimal inhibitory concentrations were6.3μg/mL and1.6μg/mL respectively.Our study developed a new strategy to express CAD by fusion with CELP tag, and highpurity CAD was obtained. Our method simplified the purification process. The resultsindicated that CELP could be applied as a powerful affinity tag for industrial-scale productionof CAD.