重组人胰岛素样生长因子1工程菌的高密度发酵(英文)

High cell-density fermentation of recombinant human insulin-like growth factor-1 engineering bacteria

背景:胰岛素样生长因子1是人体内重要的细胞调控因子,已用于治疗糖尿病等多种疾病,工程菌发酵生产工艺研究是胰岛素样生长因子1实现产业化从而扩大临床应用的关键因素。目的:探讨表达重组人胰岛素样生长因子1工程菌的高密度发酵与表达条件。设计、时间及地点:酶基因工程实验,于2007-05/2008-05在浙江树人大学生物技术实验室完成。材料:重组人胰岛素样生长因子1大肠杆菌表达菌株E.coliBL21(DE3)/pET22a-IGF-1由浙江树人大学生物技术实验室保存。高密度发酵补料培养基为:葡萄糖300g/L,蛋白胨40g/L,酵母粉10g/L,Na2HPO4280mmol/L,NaH2PO4·2H2O120mmol/L,MgSO410mmol/L,氨苄青霉素100mg/L。方法:菌株活化后,进行摇瓶发酵试验,分别改变培养基种类、诱导剂异丙基硫代半乳糖苷浓度、诱导时间等参数,优化摇瓶发酵条件。依据摇瓶发酵优化条件,采用自控5L发酵罐进行分批补料发酵试验。培养分分批培养和分批补料2个阶段。采用pH反馈流加的方式补料,在对数生长中后期开始诱导,加入异丙基硫代半乳糖苷至设计终浓度,培养4～6h。主要观察指标:重组人胰岛素样生长因子1工程菌的发酵与产物表达情况。菌体浓度与菌体干重测定,目的蛋白表达量的测定,发酵液葡萄糖浓度的测定。结果:以2×YT+5g/L葡萄糖为发酵培养基,经0.8mmol/L异丙基硫代半乳糖苷诱导5h,通过控制溶解氧以及pH反馈补料方式,实现工程菌高密度发酵与目的蛋白高效表达,每升发酵液收获干菌体50.1g/L,重组人胰岛素样生长因子1含量达5.25g/L。结论:实验成功建立了重组人胰岛素样生长因子1工程菌优化的高密度发酵工艺,为重组人胰岛素样生长因子1的下游纯化和工业化生产奠定了基础。

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. colistrain 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）, NaH2PO4.2H2O （120 mmol/L）, MgSO4 （10 mmol/L）, and ampicillin （100 mg/L）. METHODS： The strains were activated and then cultured in orbital shakers. Parameters such as types of media, isopropyl-β-D- thiogalactopyranoside （IPTG） concentration and induction time have been analyzed to explore optimal fermentation conditions for expressing the recombinant protein. According to the optimal fermentation condition of orbital shakers, batch fermentation was carried on with 5 L-autocontrol fermentor. The process contained two stages： batch culture and fed-batch through pH-stat solution. IPTG was added to induce the expression of protein in the middle and latter of the logarithmic growth phase for 4-6 hours. 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 2xYT＋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 established conditions, 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 bacteria is the useful bases for further purification and large scale production of IGF-1.