重组大肠杆菌表达的Ber e 1纯化工艺中去除内毒素效果研究

Removal of endotoxin in purification process of recombinant Ber e 1 expressed in Escherichia coli

目的去除重组巴西坚果过敏原蛋白Ber e 1(r Ber e 1)溶液中的内毒素,为后续实验和重组过敏原蛋白的临床应用打下基础。方法采用亲和层析法、超滤法和离子交换法联合应用以及Triton X-114萃取法与亲和层析法联合应用,比较这两个方案去除重组蛋白溶液中的内毒素效果。结果亲和层析法、超滤法和离子交换法联合应用的内毒素去除率为99.99%,内毒素浓度为6.25 EU/ml,蛋白回收率为42.8%;Triton X-114萃取法与亲和层析法联合应用的内毒素去除率为99.99%,内毒素浓度为2.09 EU/ml,蛋白回收率为26.1%。结论 Triton X-114萃取法与亲和层析法联合应用蛋白溶液内毒素的去除效果较佳,且内毒素含量以及Triton X-114残留量均在《中国药典》的规定范围内。

Objective To remove the endotoxin from the recombinant protein solution of Brazil nut allergen Ber e 1 to facilitate the further experimental and clinical application. Methods Two purification pathways, the combination of affinity chromatography, ultrafiltration and ion exchange chromatography, and joint applications of Triton X-l14 phase separation and affinity chromatography, were adopted to remove the endotoxin from protein solution. Results Both pathways worked well but with different efficiency. Removal rate of endotoxin was 99.99% and endotoxin level was 6.35 EU / ml with the protein recovery rate of 42.8% after affinity chromatography combined with ultrafiltration and ionexchange chromatography method. After going through Triton X-l14 phase separation and affinity chromatography method,the removal rate of endotoxin was also 99.99%, and the endotoxin concentration could be reduced to 2.09 EU / m L, with the recovery rate of 26.1%. Conclusion Triton X-114 phase separation combined with affinity chromatography method is a better method to purify the recombinant proteins and remove endotoxin. Both endotoxin and Triton X-114 residues in the purified recombinant protein are well below the required limits of Chinese Pharmacopoeia.