ABSTRACT
BACKGROUND:Insulin-like growth factor-1(IGF-1)is an important cell factor which plays a special role in many disease treatments such as diabetes.The research of engineering bacteria fermentation production technology is great to IGF-1 industrialization and to clinical application.OBJECTIVE:To investigate the high cell-density fermentation and expression condition of recombinant human insulin-like growth factor-1(IGF-1) engineering bacteria.DESIGN,TIME AND SETTING:The enzyme,gene engineering,study was performed at the Biotechnological Laboratory of Zhejiang Shuren University from May 2007 to May 2008.MATERIALS:Recombinant E coli strain for IGF-1 expression as BL21 (DE3)/pET22a-IGF-1 was reserved in the Biotechnological Laboratory of Zhejiang Shuren University.Nutrient feed was composed of:glucose(300 g/L),peptone(40 g/L),yeast powder(10 g/L),Na2HPO4(280 mmol/L),Na2HPO4-2H2O2(120 mmol/L),MgSO4(10 mmol/L),and ampicillin(100 mg/L).METHODS:The strains were activated and then cultured in orbitaI shakers.Parameters such as types of media,isopropyl-β-D-thiogalactopyranoside(IPTG)concentration and induction time have been analyzed to explore optimaI fermentation conditions for expressing the recombinant protein.According to the optimal fermentation condition of orbitaI shakers.batch fermentation was carried on with 5 L-autocontroI fermentor.The process contained two stages:batch culture and fed-bafch through pH-stat solution.JPTG was added to jnduce the expression of protein in the middle and latter of the logarithmic growth phase for 4-βhours.MAIN OUTCOME MEASURES:Following described parameters were measured:fermentation and protein expression of the recombinant human IGF-1 engineering bacteria,cell concentration,cell dry weigh,objective protein,glucose concentration.RESULTS:Cells were cultured in 2×YT+5 g,L glucose medium,induced by 0.8 mmol/L IPTG for 5 hours.By controlling dissolved oxygen and by pH-stat feeding solution,high cell-density and high protein expression were achieved.Under the establishedconditions.50.1 g dry cell weight(DCW)/L could be obtained,and 5.25 g/L IGF-1 was achieved.CONCLUSION:The established high cell-density fermentative procedure of recombinant human IGF-1 engineering bacteda is the useful bases for further pudfication and large scale production of IGF-1.