Studies On The Production Of Angiostatin By Fermentation Of Recombinant Escherichia Coli

The optimizations of culture conditions and medium constituents were carried out for the production of recombinant human angiostatin (rhAGN) by E.coli M15 (pQE32-AGN). The solution and renaturation of inclusion bodies were also studied. The influence of different culture conditions on the production of rhAGN by E.coli M15 (pQE32-AGN) were studied. The optimal conditions were as follows: 20 mL operational volume in 250 mL shake flask with inoculum 5 %; the optimal temperature was 37 oC while the initial pH was 7.0; the best induction was started when OD600 reached to 0.8～1.0 by adding 0.1 mmol/L IPTG and continued culturing for 6 h. The mass of recombinant protein expression was 35 % of the total cellular protein, and the yield of rhAGN amounted to 560 mg/L.An efficient fermentation medium for high biomass yields has been developed by single factor experiment and Orthogonal Design. The optimal levels for the constituents were: glucose 1 %,(NH4)2SO4 0.4 %,NaCl 0.2 %, KH2PO4 0.3 %,K2HPO4 2 %,MgSO4 0.01 %,VitaminB1 0.4 mg/L,VitaminB2 0.1 mg/L,VitaminB6 0.6 mg/L,D-Pantothenic acid 0.3 ml/L,FeSO4 30 mg/L,Li2SO4 30 mg/L,MnSO4 25 mg/L,Zn(CH3COO)2 20 mg/L,Al2(SO4)3 9 mg/L. When the strain was cultivated in a 5 L fermenter,dry cell weight increased to 8.06 g/L from 3.12 g/L. The high-cell density growth of engineering strain has no negative effect on the expression of target protein, and the production of rhAGN was 2.51 g/L.Conditions of cell sonication and solution of inclusion bodies were studied. The work time of ultrasonication was 10 min under 400 power and 80 mg cell per mL buffer. The inclusion bodies were solubilized in 8 mol/L urea containing 20 mmol/L DTT, 50 mmol/L Tris-HCl (pH 8.0) buffer. Then dilution, dialysis and chromatography were carried out for renaturation of rhAGN. These experiments revealed that pH has evident effect on renaturation while purity of target protein does not influence renaturation. Gel filtration chromatography was feasible to renaturation of rhAGN compared with dialysis, ion-exchange chromatography and hydrophobic chromatography. Growth inhibition rate of renaturation protein by GFC on HEMC amounted to 50.10 % under 4.28 ?g/mL. The total recovery of active protein was 55.15 %. Furthermore target protein was well purified while renaturing by GFC.