Fusion Expression Of PekⅡ Gene In Escherichia Coli And Its Primary Purification

Antimicrobial peptides(AMPs), a family of short-chain peptides with antibacterial activity, AMPs are important defense substances in innate immune system. Except for broad-spectrum antibacterial activity, AMPs have more biological activities on the resistance of virus, fungi, parasite and tumor, but scarcely induce drμg resistance, which defines a great application prospect.The contents of this studies include: Firstly, Using the preferential condon of E.coli, the novel antibacterial peptide PekⅡand pleurocidin (23~30 amino acid coding segment in N terminal) and misgurin (7~21 amino acid coding segment in N terminal), were designed and synthesized. After cheked the result of Synthesis by sequence, We have cloned the gene into 3' of the gene of Thioredoxin in the Thio-Fusion Expression Vector(pGEX-4T-2), and the fusion protein of GST-PekⅡwas highly expressed in soluble form. The specific protein brand was showed at 29 kD by SDS-PAGE. By MALDI-TOF MS, the purified protein showed a brand with 29553.03 Da. Because of the cleavage site of Enterokinase was designed in the middle of T hioredoxin and PekⅡ, the expressed Peptides of theMutation of PekⅡCould be Cuted down from the fusion Protein by Enterokinase. Secondly, The optimized induction condition was established that IPTG were added to the final concentration of 0.8 mmol/L when the bacterial cells density reached OD=0.8, then continuously culture the cells for 4h at 37℃, so that the soluble proteins GST- PekⅡcan attain a high expresstion level. Thirdly, the results of Western-blotting analysis showed that the GST-PekⅡfusion protein was successfully expressed and both the bacterial sample with m-Mdde/pGEX-4T-2 after induction by IPTG and the recombinant bacterial sample after induction by IPTG can specificially identify anti-GST antibody and the latter is bigger than the former. The bacterial sample before induction by IPTG has a empty brand. Finally, the GST-PekⅡfusion protein was purified by affinity chromatography with Glutathione SepharoseTM 4 and the GST-PekⅡfusion protein has inhibition activity to the growth of E.coli DH5αestimated by liquid growth inhibition assay.The results of this studies showed that we could produce the PekⅡsoluble expression in E.coli and this was key step to produce PekⅡlargely by fermentation. Meanwhile, the conclusion laid the foundation for the GST-PekⅡfusion protein which was partially cleaved by recombiant enterokinase, further purity and liquid growth inhibition assay.