基因工程人β干扰素的原核表达及纯化研究

Prokaryotic Expression and Purification of Recombinant Human IFN-beta

目的 :摸索一种适合于中试生产的点突变重组人 β干扰素 ( 17Ser IFN β)发酵与纯化工艺。 方法 :研究不同培养基和热诱导时间对17Ser IFN β表达的影响 ;观察pH、蛋白浓度和层析条件对17Ser IFN β纯化的影响。 结果 :17Ser IFN β工程菌在含甘油、酪蛋白水解物及微量元素的M9培养基中 ,17Ser IFN β表达量明显增加。灰度扫描显示热诱导 4h后的表达量最高 ,目的蛋白可达菌体总蛋白的 2 0 %以上。17Ser IFN β可以相对特异性地被仲丁醇抽提至有机相中 ,纯度达 80 % ,经S2 0 0凝胶过滤柱层析后纯度达 90 % ,用G2 5柱转换缓冲液 ,使缓冲液pH值 >8,同时去除缓冲液中的还原剂DTT ,将复性后的样品用C4反相柱层析进行精制 ,最终获得纯度 >98%和比活性 >3× 10 7U/mg的17Ser IFN β制品。 结论 :成功地建立了注射用新型重组人 β 干扰素的中试生产工艺 ,为随后进行的临床前研究及临床试验打下基础。

Objective: To set up a optimal method of fermentation, refolding and purification of human interferon beta ( 17 Ser IFN β). Methods: The effect of different culture medium and induction time by thermal on the contents of 17 Ser IFN β were observed The optimal condition for purification of 17 Ser IFN β was also studied by changing pH, concentration of protein and chromatography procedure. Results: The expression level of 17 Ser IFN β increased significantly in M9 culture medium containing glycerol, casein hydrolysate and trace element. The optimal induction time for 17 Ser IFN β expression was 4hr and the expression quantity could reach 20% of total protein under the condition. The soluble IFN β in SDS could be extracted into organic phase by 2 eutanol and the purity could reach 80%. High molecule weight protein was excluded from 17 Ser IFN β after S200 gel permision chromatography and the purity could reach over 90%. The buffer was converted to that with pH>8 by Superdex G25. After refolding, the product was refined by C4 reverse phase chromatography with a final purity over 98% and the biological activity more than 3×10 7 U/mg. Conclusion: The optimal condition of recombinant human 17 Ser IFN β was successfully established, which can be used for scale up production in the future.