摘要
采用两性离子胶体沉淀和乙醇沉淀相结合的粗提方法,经离子交换、疏水层析、亲和层析及凝胶过滤 4个步骤有效地将人尿激肽原酶(hk 1)粗提物纯化,比活提高了 175 5倍,总得率为 70 %.用以慈菇蛋白酶抑制剂为配体的亲和层析纯化hk 1,效果理想,整个工艺路线适合产业化生产.纯化产物在SDS 聚丙烯酰胺凝胶电泳上为单带,高压液相色谱(HPLC)上为单峰,基质辅助激光解析电离飞行时间质谱测得分子质量为 3345 0u,等电聚焦测得pI在 4 3附近,为含糖蛋白.同时测定了该酶的热稳定性和pH稳定性.纯化过程中同时分离得到另一种药用蛋白———人尿胰蛋白酶抑制剂(HUTI).
Human urinary kallikrein (hk-1) and human urinary trypsin inhibitor (hUTI) are two acidic proteins in the urine. Both of them are important drugs. The way of colloid adsorption together with ethyl alcohol precipitation isolated these two proteins successfully. By using of ion-exchange, hydrophobic, affinity chromatography and gel filtration, human urinary kallikrein (hk-1) was purified with single band on SDS-PAGE and single peak on HPLC. The molecular mass was 33 450 u on MALDI-TOF-MS, the pI was about 4.3 on IEF. The influence of pH and temperature on the activity of hk-l was studied. The whole procedure is suitable for large-scale production.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2003年第5期772-777,共6页
Progress In Biochemistry and Biophysics
基金
国家高技术(863)计划资助项目(Z18 0 3 2 9)~~
关键词
人尿激肽原酶
纯化
鉴定
抑制剂
乙醇沉淀
胶体沉淀
离子交换
疏水层析
亲和层析
human urinary kallikrein ( hk-1)
human urinary trypsin inhibitor
ethyl alcohol precipitation
colloid precipitation
ion-exchange chromatography
hydrophobic chromatography
affinity chromatography
protease inhibitor from Arrowhead (API)
