摘要
目的优化重组白喉毒素无毒突变体CRM197(recombinant cross reacting material 197,rCRM197)柱复性及纯化工艺。方法将Sephacryl S-200凝胶柱复性和Q Sepharose FF强阴离子交换层析结合,复性和纯化rCRM197;通过正交试验L9(34)考察凝胶层析复性时流速、离子交换层析纯化的pH值、淋洗液中NaCl浓度和洗脱液中NaCl浓度4种因素对rCRM197纯度及回收率的影响,筛选rCRM197复性及纯化的最佳工艺。结果包涵体经Sephacryl S-200凝胶柱复性后,相对分子质量小于36 000的杂质已被去除;经Q Sepharose FF离子交换层析后,rCRM197纯度达95%以上;在复性时流速为10 ml/min、离子交换层析纯化的pH值为7.5、淋洗液为50 mmol/L Tris-Cl+30 mmol/L NaCl、洗脱液为50 mmol/L Tris-Cl+50 mmol/L NaCl的条件下,rCRM197能达到纯度和回收率的共平衡。结论已筛选出rCRM197复性及纯化的最佳工艺,该工艺操作简便,制备的rCRM197纯度高,适用于rCRM197的产业化。
Objective To optimize the procedure for column refolding and purification of recombinant nontoxic diphtheria toxin mutant CRM197（rCRM197）. Methods The rCRM197was refolded and purified by Sephacryl S-200 gel column chromatography combined with Q Sepharose FF strong anion exchange chromatography. The effects of four factors,including refolding flow rate,pH value of purification buffer as well as sodium chloride concentrations in wash buffer and elution buffer,on the purity and recovery rate of rCRM197were evaluated by orthogonal experiment[L9（34）],based on which the optimal procedure for refolding and purification of rCRM197was developed. Results The foreign matters with relative molecular masses of less than 36 000 in inclusion body after being refolded by Sephacryl S-200 gel column chromatography were removed. The purity of rCRM197after purification by Q Sepharose FF strong anion exchange chromatography was more than 95%. The optimal refolding flow rate,pH value of purification buffer,wash buffer and elution buffer were10 ml / min,7. 5,50 mmol / L Tris-Cl ＋ 30 mmol / L NaCl and 50 mmol / L Tris-Cl ＋ 50 mmol / L NaCl respectively,under which the co-equilibration of purity and recovery rate was achieved. Conclusion The procedure for refolding and purification of rCRM197was optimized,which was easy to handle,and the prepared rCRM197showed high purity. The procedure was suitable for the large-scale production of rCRM197.
出处
《中国生物制品学杂志》
CAS
CSCD
2014年第7期945-949,共5页
Chinese Journal of Biologicals
关键词
白喉毒素无毒突变体
柱复性
纯化
正交试验
Nontoxic diphtheria toxin mutant Column refolding Purification Orthogonal experiment
