摘要
针对由中国仓鼠卵巢细胞(CHO)表达的多聚亚基蛋白HBsAg在离子交换层析过程中容易因亚基解离而导致蛋白解聚和丧失生物活性的难题,实验中选择聚乙二醇(PEG)作为保护剂伴随式(Polyethylene Glycol-Accompanied)离子交换层析分离纯化HBsAg。实验表明,在流动相中加入1%PEG10000(WV)作为纯化伴侣,HBsAg的回收率由55%左右提高到80%以上,纯化倍数基本保持在12左右。对纯化产物进行SDS_PAGE分析表明,1%PEG10000的纯化伴侣伴随式离子交换层析能全部保留HBsAg的糖基化蛋白单体(27kD和30kD),高效液相色谱联用多角度激光散射(High Performance Size Exclusion Chromatography-Multiangle Laser Light Scattering,HPSEC-MALLS)进一步分析阐明了PEG能促使HBsAg颗粒尺寸分布更均一,结构更接近天然乙肝表面抗原。
The dissociation of virus-like particles of Hepatitis B surface antigen （HBsAg） during the adsorption-desorption on the solid-phase of chromatography is a main challenge for its purification. Herein, poly （ethylene glycol） （PEG） was applied as an additive during the purification of HBsAg from recombinant Chinese hamster ovary （CHO） cell culture to improve the HBsAg recovery and protect its structural assembly. The presence of 1% of PEG10000 in the mobile phase of ion-exchange chromatography （IEC） of DEAE-Sepharose FF column could increase the recovery of HBsAg from about 55% to 80%, with a similar purification（-fold） （about 12） compared with the absence of PEG. Importantly, glycosylated protein forms of HBsAg were reserved well by PEG-accompanied chromatography. Furthermore, size exclusion chromatography-multiangle laser light scattering （SEC-MALLS） analysis was performed on line to monitor the aggregates, particle size and molecular weight distribution of HBsAg. The results demonstrated that the HBsAg particle size and assembly are more homogenous after adding PEG in the mobile phase of IEC than no PEG added in the mobile phase.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第6期947-953,共7页
Chinese Journal of Biotechnology
基金
国家高技术研究与发展计划(863计划)基金资助项目(No.2002AA217031)。~~
关键词
纯化伴侣
聚乙二醇
乙肝表面抗原
离子交换层析
recombinant Hepatitis B surface antigen （rHBsAg）, purification chaperon, ion-exchange chromatography, poly（ethylene glycol） （PEG）
