The Prokaryotic Expression, Purification And Function Research Of The Fusion Polypeptide HEGF-AWRK6

AWRK6(SWVGKHGKKFGLKKHKKH) is a new type of antimicrobialpeptides derived from Dybowskin-2CDYa (SAVGRHGRRFGLRKHRKH) andcomposed of18amino acids, molecular weight of2130.5, isoelectric point of10.78,instability coefficient of-6.74. AWRK6had little similarities with other frogsantimicrobial peptides ever discovered, it is rich in lysine and had the characteristicsof strong hydrophilic, high stability, strong antibacterial activity and broadantibacterial spectrum, these advantages make it potential to become a new type ofpeptide antibiotics.Human epidermal growth factor(hEGF) is a member of somatomedin familywhich can powerfully promote cell proliferation, it is the mitogen of sensitive cellslike epidermis cells and mesenchymal cells. hEGF has been widely used in skinwounds and burns.We obtained stable expression of the fusion polypeptide hEGF-AWRK6in E.coli expression system by IPTG induction, and studied its antibacterial properties andhealing accelerating function both in vitro and in vivo. This issue lay a foundationfor further research on other biological functions of fusion polypeptidehEGF-AWRK6.We designed and synthesized the gene sequences of hEGF and AWRK6according to the codon preference of E.coli and connected it with pET-30a(+) toconstruct recombinant plasmid pET-30a(+)-hEGF-AWRK6. After PCR, restrictionenzyme digestion and sequencing validation, transformed it into E. coli BL21(DE3)and induced it to express the target gene, confirmed the expression of fusion proteinby Western Blot. We studied the optimum fermentation condition from three aspectsincluding time, temperature and IPTG concentration. Thallus were broken byultrasonication, then collect the supernatant and the precipitation to determine theexpression form of the fusion polypeptide. The collected inclusion bodies werewashed, dissolved, renatured and purified by Nickel ions metal chelation affinitychromatography, we obtained a single component of the fusion polypeptide with His-tag after desalting and freeze-dried, remove His tag with TEV protease andhEGF-AWRK6was obtained through a secondary nickel column. Usingcolony counting method to detect the antibacterial activity, MTT and cell scratchmethod to assay cell proliferation activity of hEGF-AWRK6. Established thescalded model of mice and detected the function of hEGF-AWRK6in vivo.Results show that the prokaryotic expression plasmid pET-30a(+)-hEGF-AWRK6was successfully constructed. After induction, the positiverecombinant strain expressed the fusion polypeptide successfully. The expressionconditions of37℃,1mmol/L IPTG and fermentation time4h were optimal. Thefusion polypeptide expressed in the form of inclusion bodies, after separation andpurification, we obtained the desired protein powder (0.05-0.1g/L). The fusionpolypeptide hEGF-AWRK6had inhibitory activity on Staphylococcus aureus andEscherichia coli O157, MTT assay and cell scratch assay showed thatwithin the range of certain concentration, hEGF-AWRK6could promote cellproliferation and migration. Experiment in vivo preliminary illustrated thathEGF-AWRK6had the function to inhibit bacterial growth at the site of an injury,improve immunity, promote wound healing. In this study we acquired a fusionpolypeptide hEGF-AWRK6with biological activity which lay a theoretical andpractical basis for further study on skin repair and drug development.