| Apidaecin is a kind of cationic peptide with 18 a mino acid residues,which has a strong inhibitory effect on Gram positive and negative bacteria.At present,the most effective means of production of antimicrobial peptides is the Genetic Engineering Technique,including Escherichia coli and Pichia pastori expression systems.It's difficult to express antimicrobial peptides because of its strong toxicity,the small molecular weight and the charged amino acids.This study attempted to use a variety of different fusion proteins to express bee antimicrobial peptides in different expression systems,and selected the most appropriate expression system with activity analysis.Fusion expression of antimicrobial peptides can increase the secretion and stability of antimicrobial peptides,but also reduce the probability of degradation.However cutting fusion tag is a key step.Currently,there are two methods to cut the label,which are enzymatic method and hydrogen bromide method.These two methods are traditionally thought of being expensive,toxic or affectting the activity of antimicrobial peptides.It is important to select the appropriate fusion tags.After reading a lot of literatures,we selected three cutting methods,including inteins,ubiquitin-like proteins and Asp-Pro peptide.1)As a fusion tag,the C ter minal of the intein can self-cleavage with a definite pH value,and separating with the target protein,which does not affect the structure of the N ter minal of the target protein.2)Ubiquitin-like protein can not only stabilize the expression of antibacterial peptide AP,making it tough to be degraded,but also has a specific protease,SUMO protease1.3)Asp-Pro peptide is a kind of acid sensitive site.The fusion protein is regulated by pH values,the Asp-Pro peptide will break down in a low pH value,releasing the antimicrobial peptides.The detection of the antibacterial activity of antimicrobial peptide is relatively complex.In order to simplify the steps of detecting activity,this study fused antibacterial peptide AP with hemicellulose hydrolase and fluorescent protein,so the screening of the positive clones becomed more convenient.Facts proved that this study did simplify the screening procedure of positive transformants after fusion with the two kinds of proteins.We obtained two recombinant fusion proteins His-SUMO-AP and His-mApple-DP-AP from fermentation broth.These proteins possessed antibacterial activity without cutting the fusion tags.This study was successfully constructed recombinant strain Ec-XynB-SDB-AP and Bs-SDB-AP.These two kinds of prokaryotic expression engineering bacteria have been used to extract the intein,and we found out the self cleavage condition of SDB,releasing the antibacterial peptide AP without affecting the structure.We also constructed recombinant strain Pp-his-SUMO-AP ?Pp-mApple-DP-AP and Sc-his-mApple-DP-AP.Eukaryotic expression engineering bacteria mainly refers to the ubiquitin like protein SUMO and red fluorescent protein mApple fusion,SUMO makes the expression of antimicrobial peptides more stable,the red fluorescent protein makes screening of positive clones more convenient.These fusion methods are very effective,not only can be used for expression of antimicrobial peptide AP,expression is still difficult to detect protein expression and provides a good idea and expression strategy.Through the comparison of these expression systems,the eukaryotic expression systems may be more suitable for the expression of antibacterial peptide AP,which showed the strongest antibacterial activity.In summary,this study explored the fusions of antimicrobial peptides in different expression systems and obtained fusion proteins with antibacterial activity.These experiments laid the foundation for the large-scale industrial production of antimicrobial peptide AP,and provided some reference experience for the identification of antibacterial activity. |
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