重组肝素酶I在大肠杆菌中的表达及纯化

High-Level Expression and Purification of Recombinant Heparinase Ⅰ in Escherichia coli

优化肝素裂解酶I在大肠杆菌E.coli BL21(DE3)/pOFX-T7-SL1中的重组表达条件,结果显示接种6 h(OD600≈2.5)后加入0.25 mmol/L IPTG,在25℃下表达8 h目标蛋白酶活达到最高值1.49×105IU/L。进一步于5 L发酵罐中扩大培养,20 h后最高酶活达到5.55×105IU/L。利用亲和层析分离目标蛋白,肝素裂解酶I的纯度可达到96%,回收率为37.58%,比酶活达到1.12×103IU/mg。肝素裂解酶I的高效表达为大规模利用酶法制备低分子肝素打下了坚实的基础。

Low molecular weight heparins have been under extensive development as the improved anticoagulant. It should be relatively inexpensive to be prepared in amounts suitable for the growing market in anticoagulant drugs. Here, a study aimed at high level expression of the recimbinant Hep I in Escherichia coli was presented. The expression vector pET-15b-HepI was transformed into E. coli BL21 （ DE3 ）/pOFX-T7-SL1. The effects of different expression conditions were systematically investigated to improve the enzymatic activity of HepI. The optimal conditions were determined as follows：cuhivation at 25℃ in MBL medium, induction at 6 h after inoculation with 0.25 mmol/L IPTG, and postinduetion expression for 8 h. Under these conditions, the volumetric enzymatic activity of Hep I reached 1.49 ×10^5 IU/L. The recombinant E. coli was further cultivated in a fermentor. As a result, the total activity of HepI reached 5.55 ×10^5 IU/L after 20 h fermentation. One affinity chromatography step was employed to obtain high-purity （96%） with a recovery ratio of 37.58%. This study indicated that effective expression of Hep I by the present system would facilitate the large scale preparation of low molecular weight heparin.