Ernst S, Venkataraman G, Winkler S, Godavarti R, Langer R, Cooney C L, Sasisekharan R
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139, USA.
Biochem J. 1996 Apr 15;315 ( Pt 2)(Pt 2):589-97. doi: 10.1042/bj3150589.
The use of heparin for extracorporeal therapies has been problematical due to haemorrhagic complications; as a consequence, heparinase I from Flavobacterium heparinum 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 Km (47 micrograms/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. heparinum. 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 hexa-saccharide 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.
由于出血并发症，肝素在体外治疗中的应用一直存在问题；因此，来自肝素黄杆菌的肝素酶I被用于测定血浆肝素以及从循环中清除肝素。在此，我们报道了重组肝素酶I在大肠杆菌中的表达、纯化至同质以及对纯化酶的特性鉴定。肝素酶I以带有N端组氨酸标签的形式表达。该酶不溶性且无活性，但可进行重折叠，并通过镍螯合色谱纯化至同质。累积产率为43%，纯化的肝素酶I回收率为每升培养物14.4毫克。通过基质辅助激光解吸质谱分析的N端序列和分子量与肝素酶I基因结构的预测结果一致。纯化的重组肝素酶I的胰蛋白酶消化产物的反相高效液相色谱图谱、米氏常数Km（47微克/毫升）和比活性（117单位/毫克）与天然酶相似。肝素酶I对肝素的降解产生特征性的产物分布，这与肝素黄杆菌的肝素酶II或III降解所获得的产物分布不同。我们开发了一种快速阴离子交换高效液相色谱方法，使用POROS灌注色谱介质分离肝素酶解产物。在10分钟内即可分离出特征性的二糖、四糖和六糖产物。这些重组肝素酶I的表达、纯化和分析方法可能有助于基于肝素酶I的医学治疗的进一步发展，以及对肝素和硫酸乙酰肝素的结构及其在细胞外基质中的作用的进一步研究。
