重组人骨形成蛋白-4的大规模制备

The Large Scale Preparation of Recombinant Human Bone Morphogenetic Protein-4 Mature Peptide

含有能够高表达重组人骨形成蛋白成熟肽(recombinant human bone morphogenetic protein 4 mature peptide,rhBMP- 4m)质粒的工程菌株,导入15 L NBS发酵罐中进行恒溶氧高密度发酵和诱导表达,测定发酵菌液OD600值为30．8。离心收集菌体, PAGE电泳后光密度扫描表明hBMP-4m占细菌总蛋白量的42％。悬浮所收集的菌体,裂菌,洗涤4次后的包涵体中hBMP-4m占蛋白量的83．2％。预先取少量样品进行探索实验后,全部包涵体用8 mol尿素缓冲液溶解,上SP-Sepharose FF阳离子柱,以0.35 mol NaCl洗脱,获得纯度为96％的hBMP-4m。其收获量为1．34g/L发酵液;收得率为36．4％。结果表明:使用这套工艺流程大规模制备hBMP-4m,能够得到较好的收得率、较高的收获量和纯度。

The engineering strain for high expressed rhBMP-4m was fermented and induced expression in a 15 L NBS automatic fermenter with an invariable oxygen solubility control. At the end of the fermentation, the OD600 of the bacterial cultures was 30.8. The resulting bacteria were collected by centrifigation. SDS-PAGE showed that the rhBMP-4m occupied 42% of the total bacterial protein. After lysis of the bateria, the inclusive body was resuspended, with the helping of ultrasonic treatment, and washed for 4 times, rhBMP-4m account for 83.2% of the proteins in the inclusive body after washing. The inclusive body were dissolved in 8 tool urea pH6.5 buffer and loaded on SP-Sepharose FF column. According to pre-experiment data, the rhBMP-4m was collected in batch 0.35 tool NaC1 elution portion. The purity of the purified rhBMP-4m was 96% and the harvest was 1.34 g/L fermentation liquid with the recovery rate of 36.35%. The results showed that the fermentation program and purification techniques could be used to prepare pure rhBMP-4m in large scale.