Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum
Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum
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DOI：
10.1042/bj3150589
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发表时间：
1996-04-15
影响因子：
4.1
通讯作者：
Sasisekharan, R
中科院分区：
文献类型：
--
作者：
Ernst, S;Venkataraman, G;Sasisekharan, R
由于出血并发症，肝素用于体外治疗存在问题;因此，来自肝素黄杆菌的肝素酶I用于测定血浆肝素和从循环中消除肝素。在这里，我们报告重组肝素酶I在大肠杆菌中的表达，纯化的同质性和纯化的酶的特性。肝素酶I与N-末端组氨酸标签一起表达。该酶不溶且无活性，但可复性，并通过镍螯合层析纯化至均一。累积产率为43%，纯化的肝素酶I的回收率为14.4 mg/l培养物。通过基质辅助激光解吸MS分析的N-末端序列和分子量与肝素酶I基因结构的预测一致。胰蛋白酶消化物的反相HPLC曲线、Michaelis-Menten常数K-m（47 μ g/ml）和纯化的重组肝素酶I的比活性（117单位/mg）与天然酶相似。肝素酶I降解肝素导致特征产物分布，其不同于用来自F的肝素酶II或III降解所获得的产物分布。肝素我们开发了一种快速阴离子交换HPLC方法，使用波罗斯灌注色谱介质分离酶促肝素降解产物。特征二，四和六糖产物的分离在10分钟内进行。这些重组肝素酶I的表达，纯化和分析的方法可能有助于进一步开发肝素酶I为基础的医学疗法，以及进一步调查肝素和硫酸乙酰肝素的结构和它们在细胞外基质中的作用。
The use of heparin for extracorporeal therapies has been problematical due to haemorrhagic complications; as a consequence, heparinase I from Flavobacterium heparimum is used for the determination of plasma heparin and for elimination of heparin from circulation. Here we report the expression of recombinant heparinase I in Escherichia coli, purification to homogeneity and characterization of the purified enzyme. Heparinase I was expressed with an N-terminal histidine tag. The enzyme was insoluble and inactive, but could be refolded, and was purified to homogeneity by nickel-chelate chromatography. The cumulative yield was 43 %, and the recovery of purified heparinase I was 14.4 mg/l of culture, The N-terminal sequence and the molecular mass as analysed by matrix-assisted laser desorption MS were consistent with predictions from the heparinase I gene structure. The reverse-phase HPLC profile of the tryptic digest, the Michaelis-Menten constant K-m (47 mu g/ml) and the specific activity (117 units/mg) of purified recombinant heparinase I were similar to those of the native enzyme. Degradation of heparin by heparinase I results in a characteristic product distribution, which is different from those obtained by degradation with heparinase II or III from F. heparimum. We developed a rapid anion-exchange HPLC method to separate the products of enzymic heparin degradation, using POROS perfusion chromatography media. Separation of characteristic di-, tetra- and hexasaccharide products is performed in 10 min. These methods for the expression, purification and analysis of recombinant heparinase I may facilitate further development of heparinase I-based medical therapies as well as further investigation of the structures of heparin and heparan sulphate and their role in the extracellular matrix.