利用重组基因工程菌制备降血压肽的工艺研究

Processing Technologies for Preparation of Antihypertensive Peptides from Recombinant E.coli BL21

摘要:

本文利用正交实验方法，对重组降血压肽基因工程菌细胞破碎所得上清液中的融合蛋白GST-AHP，进行亲和层析吸附上柱条件研究。在此基础上，借助高效液相色谱对融合蛋白凝血酶酶切条件进行了研究，并对此分离条件下所得的降血压肽段的体外生物学活性进行初步确定。实验表明，基因工程菌细胞破碎上清液中的融合蛋白GST-AHP进行亲和吸附的最佳上柱条件为上柱量为4 mg/mL，上柱流速为1.5 mL/min，上柱1次，此条件下融合蛋白的吸附量达3.86 mg/mL，洗脱后其回收率达96.5%；凝血酶酶切的最佳工艺条件为1 mg GST-AHP融合蛋白添加凝血酶15 U，22 ℃酶切8 h，此条件下可得降血压肽段102 μg。分离纯化所得降血压肽段的体外生物学活性良好。

Abstract:

The preparation of AHP from recombinant E.coli BL21 by affinity chromatography and thrombin mediated cleavage was studied through orthogonal experiment. RP-HPLC was used to detect the antihypertensive peptides in AHP. The optimum loading quantity of sample and flow rate were 4 mg (GST-AHP)/mL (medium bed) and 1.5 mL/min, respectively, under which, the absorption capacity of fusion protein was 3.86 mg/mL (medium bed) with a recovery rate of 96.5%. The optimum cleavage conditions of fusion protein by thrombin were as follows: thrombin dosage of 15 U /mg GST, reaction time of 8 h and temperature of 22 ℃, under which 102 μg of AHP could be obtained using 1 mg GST-AHP. In addition, bioactivity of recombinant AHP was studied. The obtained AHP showed high bioactivity in vitro with IC50 and Km of 4.6 μmol/L and 1.95 mmol/L, respectively.