家蚕丝素-RGD融合蛋白的盐析纯化工艺探讨

利用基因重组技术合成了具有良好细胞粘附活性的重组家蚕丝素-RGD融合蛋白质(Silk-RGD),探讨了其盐析的工艺条件。研究发现,在融合蛋白等电点pH8附近,当硫酸铵在溶液中的饱和度较低(10%)时,其蛋白质沉淀量随着温度的升高而增加;当硫酸铵的饱和度升至20%时,蛋白质沉淀量不再随着温度的变化而变化,但与低饱和度硫酸铵时相比,沉淀量明显增加;当硫酸铵的饱和度升至30%或以上时,沉淀中杂蛋白含量明显增多,不利于目的蛋白的纯化。Silk-RGD融合蛋白相对合理而且易操作的盐析纯化条件为:pH8,硫酸铵在溶液中的饱和度为20%,室温(约20℃)。利用该工艺条件,可以低成本、高效率地生产Silk-RGD融合蛋白,从而满足新型材料规模化开发的需要。

Analysis on Salting-out Purification of the Bombyx mori Silk Fibroin-RGD Fusion Protein

By applying of genetic engineering strategies a Bombyx mori silk fibroin-RGD fusion protein(Silk-RGD) was synthesize,which showed a high cell adhesive ability,and the technological conditions for salting-out purification of the Silk-RGD were studied.The results revealed that the yield of salting-out precipitate increased with rise of temperature at the low(NH4)2SO4 concentration(10%) under pH 8 of isoelectric point;when the(NH4)2SO4 concentration increased to 20%,the yield was not temperature-dependent but much more than that at concentration of 10%;when the(NH4)2SO4 concentration increased to 30% or above,there were many impurities in the precipitates causing difficulties for purification of the target protein.As a conclusion,it seemed that the rational and easy operating salting-out conditions for purification of Silk-RGD were 20% of(NH4)2SO4 at room temperature(around 20 ℃) and pH 8.Under these conditions,Silk-RGD can be produced with high efficiency and low cost to meet the demands of development of new material in large scale.