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目的探索建立1种串联离子交换层析和固相化金属螯合亲和层析从Cohn法组份Ⅳ分离纯化蛋白C的方法 ,降低纯化蛋白C的成本,并实现血浆的综合利用。方法 4℃条件下,将Cohn法组份Ⅳ用PBS缓冲液抽提5h,离心(6500g)40min,上清液依次用0.45μm和0.22μm的滤膜过滤后,超滤透析;再通过串联离子交换层析和固相化金属螯合亲和层析,对蛋白C进行纯化。通过SDS-PAGE鉴定蛋白C的纯度;用考马斯亮蓝法通过紫外分光光度计测定总蛋白含量;通过WHO指定的发色底物法和凝固法(一期法)测定蛋白C活性。结果纯化的蛋白C在SDS-PAGE图谱上呈现1条62kD左右的蛋白带;经过离子交换层析,蛋白C的活性回收率为(48.19±9.22)%,纯化倍数为(3.75±0.56)倍;固相化金属螯合亲和层析后,蛋白C活性回收率为(59.61±5.59)%,纯化倍数为(36.79±3.72)倍;蛋白C总活性回收率为(28.77±9.45)%,纯化倍数为(136.64±6.82)倍,比活性为(11.70±0.89)IU/mg。结论串联离子交换层析和固相化金属螯合亲和层析可以从Cohn法组份Ⅳ中获得比活性较高的蛋白C浓缩物。
OBJECTIVE: To establish a method for the isolation and purification of protein C from Cohn fraction Ⅳ by tandem ion-exchange chromatography and solid-phase metal chelate affinity chromatography, to reduce the cost of purified protein C and to achieve the comprehensive utilization of plasma. Methods The Cohn fraction IV was extracted with PBS buffer for 5 h at 4 ° C, centrifuged (6500 g) for 40 min, and the supernatant was filtered through 0.45 μm and 0.22 μm filters followed by ultrafiltration and dialysis. Protein C was purified by exchange chromatography and solid phase metal chelate affinity chromatography. The purity of protein C was identified by SDS-PAGE. Total protein content was determined by UV spectrophotometer using Coomassie Brilliant Blue method. The protein C activity was determined by WHO chromogenic substrate method and coagulation method (first phase method). Results The purified protein C showed a protein band of about 62 kD on SDS-PAGE. The recovery rate of protein C was (48.19 ± 9.22)% and the purification fold was (3.75 ± 0.56) fold after ion exchange chromatography. After immobilized metal chelate affinity chromatography, the recovery rate of protein C activity was (59.61 ± 5.59)% and the purification fold was (36.79 ± 3.72) times. The total recovery of protein C was (28.77 ± 9.45)%, Times (136.64 ± 6.82) times, specific activity (11.70 ± 0.89) IU / mg. Conclusion Tandem ion exchange chromatography and immobilized metal chelate affinity chromatography can obtain more concentrated protein C concentrates from Cohn method.