Expression,Purification And Function Of The HEGF-Dybowskin-2CDYa Fusion Polypeptide

Dybowskin-2CDYa(accession no.:EU827809)is a kind of cationic peptide which is separated and named by our laboratory from Rana dybowskii and it was rich in Arginine(SAVGRHGRRFGLRKHRKH)and relative molecular mass was 2154.2 with isoelectric point of 12.60Dybowskin-2CDYa is a new type peptide which has more positive charge,higher isoelectric point and strong hydrophilicity.In our preliminary study,it showed that Dybowskin-2CDYa has higher bacteriostatic activity and weaker hemolytic activity.It is concluded that the peptide shows potential for development into a therapeutically valuable agent as antibioticdrug.Human epidermal growth factor(hEGF)is a member of somatomedin family which can powerfully facilitate cell proliferation.hEGF fusion protein has remarkable effect on scald,burn,corneal wound and gastric ulcer.It can also work on neovascularization,proliferation of epidermis cell and differentiation of nerve cells.Therefore,AEGF can treat diseases as wound,burn and healing acceleration.In the preliminary tests,we obtained the fusion gene sequence and constructed a recombinant plasmid pET30-hEGF-Dybowskin-2CDYa.The recombinant protein was successfully expressed by inducing with IPTG.In this article we optimized the expression condition to purified the peptide and carried on some function studies.The level of expression was influenced by the constitution of culture medium,temperature,pH,the concentration of IPTG,induction time and ventilatory capacity.We focus on the induction by time,pH and temperature to optimize the expression condition.After centrifugation,the supernatant was discarded.Then ultrasonication,the supernatant and the precipitation was collected by centrifugation(3000rpm,10min)and analyzed by 12%SDS-PAGE.It showed that hEGF-Dybowskin-2CDYa also expressed as an inclusion body even in the low temperature.Affinity purification of this fusion peptide was labeled by His-tag.To ensure the bioactivity of fusion peptide in vacuum freeze-drying,we added a protective agent into it.The optimized regent was selected by redissolving detection of bacteriostatic activity with MIC.Measure the activity of hEGF by MTT assay and cell scratch.The result shows that the optimized fermentation conditions was at 37?,lmmol/ml(IPTG)for 4 hours.Average of 1 Liter fermentation broth would collect 7g thallus and 0.2-0.3g protein powder.The purified protein after refolding proceeded bacteriostatic test showing it had high activity against gram-positive and gram-negative bacterium.The MIC of Escherichia coli,Pseudomonas aeruginosa and staphylococcus aureus was carried on respectively.MTT assay and cell scratch showed that hEGF-Dybowskin-2CDYa had effect on cell proliferation and migration in a concentration-dependent manner.This study lays a material foundation on the structure and function of Dybowskin-2CDYa and its inhibition mechanism and a theoretical basis for its subsequent study on skin-repair and new drug development.