Construction Of An Efficient Bacillus System For Extracellular Production Of The Human Epidermal Growth Factor

since1970s,Bacillus expression system obtain many achievements in theapplication,it is cloned and expressed many prokaryotic and eukaryoticgenes.However,due to the use of Bacillus on expression of exogenousproteins is far behind E.coli,Pichia pastoris which has been maturelycommercialized,the Bacillus expression systems also exist extracellularprotease degradation of exogenous proteins and genetically engineeredbacteria bottlenecks such as poor genetic stability,will be used toproduce biological protein from Bacillus product technology is stillconcentrated in a few companies and individuals,and therefore researchon Bacillus expression system has great value.EGF(Epidermal growth factor) is a potent factor in promoting celldivision and exert biological effects in epithelial cells,mesothelialcells and endothelial cells.It stimulate skin,cornea,lung,trachealepithelium in vivo growth and reproduction to accelerate the repair ofcorneal、inhibition of gastric acid secretion,increasing protein、RNAsynthesis and cell metabolism of epidermal cells and induced by neonataleyelids open.It have curative effect for certain cancers.In addition,EGF can be used in tissue culture as a biologically active additives,andit is an important raw material for cosmetics.With in-depth features andsafety aspects related to the study, making it in medicine,cosmetics andother industries with good development prospects.The purpose of this study was to explore the use of Bacillus subtilisand Bacillus brevis as a system to express human epidermal growth factorgene.In this paper,for the purpose of express hEGF gene in Bacillussubtils,the vector p05-gE using Pgrac promoter and Bacillus subtilislevansucrase signal peptide to regulate the expression of human epidermalgrowth factor gene was constructed and were transformed into Bacillussubtilis SCK6;and in Bacillus brevi,the multiple and tandemly arrangedpromoters and signal peptide of the cell wall protein of Bacillus breviswas used to regulate the expression by the vector pNY-cwpE,and Bacillusbrevis HPD31-SP3was used as the host.After the shake flask experimentsand SDS-PAGE electrophoresis,the expression of results were detected intwo strains of the culture supernatant of recombinant bacteria.This paper also of brevibacillus brevis engineering bacteriafermentation conditions explored, with soluble starch as carbon source,with peptone and yeast extract as the nitrogen source feeding composition,in a40L fermentor scale after48h fermentation cycle, the final cellwet weight can reach80g/L, the expression amount of the target proteinwas about200mg/L. Then we use the CM Sepharose F.F gel chromatographyon fermentation product containing the target protein was purified byion exchange step, and detected the proliferation activity ofpurification of the expressed product of Balb/c3T3cells.