重组人抗血栓蛋白(rHAP)工程菌表达条件的优化

Optimization of Cultivation Conditions of Recombinant Escherichia Coli Strain Expressing Human Anticoagulant Protein (rHAP)

优化重组人抗血栓蛋白(rHAP)工程菌的培养基及培养和表达条件,提高菌体产量及可溶性的重组rHAP蛋白含量。通过单因素及正交实验,对影响重组大肠杆菌生长及rHAP蛋白表达的培养基的组分、诱导剂的浓度、培养温度等工艺条件进行优化。结果表明半合成培养基成分的最优配比:0.5%蔗糖、1%蛋白胨、3%酵母提取物,IPTG诱导剂浓度为0.2 mmol/L,37℃培养4 h,开始诱导5 h,诱导温度为32℃。优化培养基及培养条件提高了菌体的生长密度及可溶性rHAP目标蛋白的表达量,表达菌密度OD600和可溶性rHAP目标蛋白表达量分别是没有优化前的1.85倍和2.59倍。在20 L发酵表达时,rHAP工程菌OD600值高达73,菌体湿量为1 957 g/20 L。

In order to increase the growth and soluble expression of recombinant E.coli strain expressing human anticoagulant protein（rHAP）,culture medium and cultivation conditions of recombinant E.coli were optimized.Single factor and orthogonal experiments were conducted to investigate the effect of cultivation conditions such as medium composition,inducer dosage,cultivation temperature etc.on the growth of recombinant bacterial strain and product of rHAP.The optimal fermentation conditions were as follows： 0.5% sucrose,1% tryptone,3% yeast extract,0.2 mmol/L IPTG,expression produce： growth at 37℃ for 4h,and induction at 32℃ for 5h.The optimized cultivation and expression condition could increase the bacterial growth density and the product of solubly expressed rHAP protein by 1.85 fold and 2.59 fold respectively,compared with the original,non-optimized condition.When fermented in 20Liter fermentor,the recombinant rHAP-expressing bacteria grew to OD600 of 73 with the wet weight of bacteria of 1957g/20L.