Design, Screening And Analysis Of Activity Of Novel Antibacterial Protein

As we all know, discovery of antibiotics is one of the significant events in our human history. Antibiotics have played a crucial role in fighting against pathogenic microorganisms. The issue of bacterial resistance, which due to the overuse of antibiotics in recent decades, has become a serious problem faced by the world. It is urgent to search for novel antimicrobial substances that can take the place of the currently used antibiotics to meet the clinical needs. Antimicrobial peptides(AMPs) and lysozymes have become the focuses of increasing attention because of their broad antimicrobial activities toward various bacteria and fungi. They play important roles in an organism’s first line of defense. They are potential alternatives for antibiotics. Many previous studies have mainly focused on the discoveries of AMPs and lysozymes and the mechanism of their antimicrobial activity. It is difficult to carry out researches on their clinical application.In the current study, 13 previously reported AMPs were expressed in Escherichia coli. A His6-SUMO tag was fused to enhance expression and offer a convenient method for purification. Fusion genes were cloned to the vector of pETDuet. Seven of them had soluble expression. We detected their yields and valued their antimicrobial activity against Gram-negative E. coli(ATCC25922) and Salmonella paratyphi(CMCC(B) 50094) as well as Gram-positive Bacillus subtilis(ATCC6633). After comparing their antimicrobial activities, Thanatin was identified to have highest yield and robust antimicrobial activity.Then we mutated Thanatin with adding arginine and asparticacid based on the antibacterial mechanism of AMPs to enhance its activity and made some single amino acids mutations of Thanatin to explore the relationships between amino acids and activity. But the mutations with more positive charge and negative charge did not enhance its activity, in contrast, antimicrobial activity became weaker as the number of amino acids at either terminus increased. And the MICs of substitution mutation of Thanatin demonstrated Val6, Ile8, Ile9 play important roles in maintaining antimicrobial activity and other mutations have little effect.Six lysozymes were selected to be expressed as well as their hybrids genes with three different antimicrobial peptides Thanatin, Moricin, Came were made respectively to expressed with His6-SUMO fusion proteins in pETDuet vector. Just T4 lysozyme, ST4 lysozyme, Thanatin-T4 L and Thanatin-ST4 L have soluble expression. T4 lysozyme, ST4 lysozyme exert excellent antimicrobial activity against Bacillus subtilis(ATCC6633), while the activity of Thanatin-T4 L and Thanatin-ST4 L against Gram-negative bacteria showed a robust decline compared to individual Thanatin and against Gram-positive bacteria showed slight decline compared to individual T4 lysozyme and ST4 lysozyme. In addition, eleven hybrids of T4L-AMPs and five Thanatin-AMPs were made and expressed. Six of T4L-AMPs have soluble expression, but their antimicrobial activity are weaker than T4 L. Four Thanatin-AMPs have soluble expression and all of the proteins degraded from their His6-SUMO tags as well their activity declined.Disulfide bond was thought to paly important roles in maintain the antimicrobial activity of Thanatin. We values the MICs of Thanatin synthesized with disulfide bond and without disulfide bond respectively. As a result, Thanatin without disulfide bond showed a better activity. We speculated the disulfide bond is not prerequisite for its structure. Then we attempted to get its crystal structure, but we failed.At last, we constructed the recombinant plasmids pHT01-His6-SUMO-Thanatin, pHT01-His6-SUMO-T4 L, pHT43-His6-SUMO-Thanatin, pHT43-His6-SUMO-T4 L to express Thanatin and T4 lysozymes in Bacillus subtilis expression system. But target fusion proteins did not been detected in the medium and bacteria by SDS-PAGE. At the same time we tried to make large-scale expression of Thanatin in E. coli and we got the wet cells of 7.84 Kg.