基因重组可溶性非融合血管生成抑制剂Kringle 5的色谱分离和纯化

Kringle 5是血纤维蛋白溶酶原中特异抑制内皮细胞增生和迁移活性最高的一种血管生成抑制剂。该实验在前期成功克隆和表达可溶性非融合血管生成抑制剂Kringle 5的基础上,建立了一种两步色谱法分离纯化Kringle 5的方法。首先用SP Sepharose Fast Flow强阳离子交换色谱柱对Kringle 5重组菌体破碎上清液进行初步分离,然后再用丙烯葡聚糖凝胶S-100 HR凝胶排阻色谱柱对其进行进一步的纯化。采用本方法得到的可溶性非融合血管生成抑制剂Kringle 5经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和高效凝胶排阻色谱检测其纯度大于98%,通过鸡胚尿囊膜法确定这种蛋白质具有抑制内皮毛细血管生长的活性。

Isolation and purification of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5 using chromatography

The Kringle 5 domain of plasminogen is one of the most potent angiogenesis inhibitors known to date,which can inhibit cell proliferation and migration efficiently.In the study,on the foundation of successful clone and expression of recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5,a two-step chromatographic method,including the use of SP Sepharose Fast Flow cation exchanger and Sephacryl S-100 HR size exclusion chromatography in sequence,was established to separate and purify angiogenesis inhibitor Kringle 5.On the SP Sepharose Fast Flow column,the buffer A consisted of 50.0 mmol/L acetic acid-sodium acetate(pH 5.2),and the buffer B consisted of buffer A with the addition of 0.5 mol/L sodium chloride(pH 5.2);on Sephacryl S-100 HR column,the elution buffer was 5.0 mmol/L phosphate solution(pH 7.0).Through the two-step chromatographic purification process,the purity of the obtained Kringle 5 was more than 98%.In addition,it was found that the obtained Kringle 5 could inhibit the blood vessel growth of chick embryo chorioallantoic membrane effectively.Finally it is concluded that this method can effectively separate active recombinant soluble and non-fusion angiogenesis inhibitor Kringle 5.