Construction Of The MMA-E Gene Encoding Mutant Of Hybrid Antibacterial Peptide And Expression In E.coli

MA-E is a synthesized hybrid antibacterial peptide with ideal activity. The design of which maintains the typical structure of cecropins: two ct-helical regions linked by a hinge region. The sequence of the NH2-terminus half of MA-E is based on the NI-12terminus of magainin, from residual amino acid 3-14. The hinge region is composed of Ala-Gly-Pro, while the COOK-terminus half is derived from NH2-terminus of melittin, from residual amino acid 3-13. MA-B gene is composed based on the amino acid sequence of MA-E. This gene has been cloned and expressed in E.coli.A site-mutation is introduced to MA-E gene by using PCR technique, and a new mMA-E gene (mutated MA-B gene) is generated. This mutated gene takes the aim to increase the ct-helical structure of magainin half and the amount of positive charge of melittin half located at the NH2 and COOK termini of MA-E respectively, which consequently strengthen the bacteriocidal activity of MA-B. mMA-E gene is 81 bp long, encoding 27 amino acid residues. The new gene is linked to the vector pTYB2 and transformed into E. coli DH5ct and ER2566 respectively. After sequencing, the recombinant plasmid PTYB2mMA~B is proved to insert sucessfully and is in accordance with the design. The recombinant strain ER2566 is induced for protein expression and the product is then purified by IMPACT-T7 One Step Protein Purification System Kit and detected by Tricne-SDS-PAGE. After silver staining, one distinct and expected band of Intein-CBD (Intein-chitin Binding Domain) is observed, with a molecular mass of 55 Kdal . A test against the purified product for its antibacterial activity is perfomed using Agar Diffusion Method, and is found that the product is bacteriocidal. These results show that mMA-E has successfully expressed in B. coli.