Expression Of Candida Tropicalis KRE9 And Its Interaction With Antibacterial Peptide CAG-N12

The antibacterial peptide CGA-N12 has specific resistance to Candida tropicalis MYA-3404 and is able to bind to the KRE9 protein by synthesized Candida tropicalis cell wall β-1,6-glucan.Therefore,the study of the interaction between the antimicrobial peptide CGA-N12 and KRE9 protein is a great significance for revealing the target of the antimicrobial peptide CGA-N12 on Candida tropicalis.First,the prokaryotic expression vector pET28a-KRE9 was constructed and transfered in E.coli BL21(DE3).The fusion protein KRE9 with a molecular weight of 35 kDa was purified by His tag purification resin after SDS-PAGE and Western Blot analysis.By measuring glucose consumption per unit time,the enzyme activity of the fusion protein KRE9 had higher enzyme activity.The interaction between the antibacterial peptide CGA-N12 and KRE9 protein was detected by isothermal titration calorimetry(ITC).The results showed that the interaction process was endothermic;the interaction force was mainly hydrophobic and little electrostatic force;the antimicrobial peptide CGA-N12 and KRE9 ratio was 6.0;and the N value was around 1,indicating that the combination of the antimicrobial peptide CGA-N12 and KRE9 as a single site binding model;the binding constant Ka value was 1489~2291,indicating a strong interaction force.In order to study the distance between the antimicrobial peptide CGA-N12 and KRE9 protein,the prokaryotic expression vectors pETDuet-YFP-CGA-N12,pETDuet-CFP-KRE9 and pETDuet-CFP-KRE9-YFP-CGA-N12 were constructed.The co-expression of the two genes in E.coli BL21(DE3)was performed with FV1000 confocal laser scanning microscopy.The interaction of the antibacterial peptide CGA-N12 with KRE9 protein was detected by fluorescence resonance energy transfer(FRET).The results showed that there was energy transfer between the antibacterial peptide CGA-N12 and KRE9 protein: the fluorescence intensity of the donor CFP-KRE9 was weakened,and the fluorescence intensity of the receptor YFP-CGA-N12 was enhanced.This showed that the distance between the antimicrobial peptide CGA-N12 and KRE9 was 7~10 nm.