基因重组人骨形成蛋白4成熟肽在大肠杆菌中的大规模发酵表达及纯化

含有编码人骨形成蛋白4成熟肽(Bone morphogenetic protein 4 mature peptide,hBMP-4m)cDNA表达质粒的菌株,经50代传代,质粒不丢失,仍然稳定高表达hBMP-4m。将此菌株导入15 L NBS发酵罐中进行恒溶氧高密度发酵、诱导表达,测定发酵菌液OD600值为43.5,离心收集菌体,PAGE电泳结果hBMP-4m占细菌总蛋白量的39％。悬浮所收集的菌体、裂菌,洗涤后的包涵体中hBMP-4m占蛋白量的85％。将少量包涵体用8 mol尿素缓冲液溶解分别上SP阳离子和Q阴离子交换柱,用连续盐浓度的洗脱液洗脱蛋白,收集各蛋白峰做蛋白电泳分析,找到洗脱液的最合适盐浓度。然后将全部包涵体用8 mol尿素缓冲液溶解,上阳离子交换柱SP柱,以最佳盐浓度洗脱液洗脱,获得纯度为91％的hBMP-4m。再上阴离子交换柱Q柱,最佳盐浓度洗脱液洗脱后,所得hBMP-4m纯度为98％。

Large Scale Fermentation, Expression and Purification of Recombinant Human Bone Morphogenetic Protein 4 Mature Peptide from Escherichia coli

First the strain that contains the mature gene of bone morphogenetic protein-4 was cultured for 50 generations and those that expressed the mature gene of bone morphogenetic protein-4 were selected as the engineering strains. The BMP-4 engineering strain was fermented in large scale in 15 L NBS fermenter and OD of bacterial culture was measured when fermentation was over. The cells were centrifuged and resuspended by STE buffer then treated with lysozyme to break the bacterial cell wall to release the pellets. After that procedure the pellets were washed by washing liquid for 4 times meanwhile treated with ultrasound each time then a little part of them was dissolved in denaturation buffer and loaded to positive ion exchange chromatography and washed by washing buffer containing (0-1) mol sodium chloride. The protein peaks containing BMP-4 were collected and the relevant sodium chloride concentration of the washing buffer was decided. Then the protein was dissolved and purified by negative ion exchange chromatography with washing buffer containing (0-1) mol sodium chloride. The objected protein peaks were collected and the relevant sodium chloride concentration of the washing buffer was decided. After that the large-scale purification of BMP-4 was done by positive and negative ion exchange chromatography with washing buffer containing decided sodium chloride concentration according to former pre-experiment BMP-4 with high purification (98%) was got.