相分离联合层析去除类人胶原蛋白中的内毒素  

Endotoxin removal from human-like collagen using Triton X-114 two-phase extraction and affinity chromatography resin

作　　者：段志广[1,2] 朱晨辉 张婧[1,2] 范代娣 

机构地区：[1]西北大学化工学院,陕西西安710069 [2]陕西省生物材料与发酵工程研究中心,陕西西安710069

出　　处：《化学工程》2017年第10期6-11,共6页Chemical Engineering(China)

基　　金：西安市教育厅专项基金(12JK0449)

摘　　要：研究类人胶原蛋白(HLC)中内毒素的去除方法,为规模化制备医用生物材料提供参考。采用Triton X-114双水相分离法作为内毒素去除工艺的第1步,亲和层析法作为工艺的第2步,二者偶联操作从类人胶原蛋白中除去内毒素,并对除去内毒素后的HLC进行了理化性能检测。结果表明:采用Triton X-114双水相分离法,在蛋白质量浓度2.06 mg/m L,作用温度44.71℃,作用时间3 h,溶液pH值为7时双水相分离系数K=9.58,蛋白中内毒素含量由10 000—25 000 EU/mg下降为1.5—2.0 EU/mg,蛋白回收率为95.6%;偶联了多聚赖氨酸亲和配基的亲和层析后,HLC中内毒素含量由1.5—2.0 EU/mg降至0.025—0.25 EU/mg,蛋白回收率可达81.9%;2步操作均不会使蛋白发生相对分子质量及化学结构的改变。此工艺操作简便,可显著降低生产成本。Triton X-114 two-phase extraction was applied as the first step to remove the large amounts of endotoxins from the preparation, and affinity chromatography resin, attached with modified polylysine, was used as the second step to achieve further purification. Furthermore, the processed protein was tested in physical and chemical properties. The results show that in the Triton X-114 two-phase extraction process, the content of HLC is 2.06 mg/ mL and the sample （ pH 7 ） is incubated at 44.71 ℃ for 3 h. Under this circumstance, endotoxin level in HLC is declined from 10 000-25 000 EU/mg to 1.5-2.0 EU/mg with high protein recovery （95.6%）. Accompanied with affinity chromatography resin, endotoxin level in HLC is declined from 1.5-2.0 EU/mg to 0. 025-0.25 EU/mg with 81.9% protein recovery. These two procedures do not change the molecular mass or chemical structure of HLC. The process is simple and convenient to carry out and can reduce the production cost significantly.

关 键 词：类人胶原蛋白 内毒素 TRITON X-114双水相分离 亲和层析 

分 类 号：TQ028[化学工程] TQ936.2