Immobilization of recombinant heparinase I fused to cellulose-binding domain.
Immobilization of recombinant heparinase I fused to cellulose-binding domain.
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DOI：
10.1002/(sici)1097-0290(19991005)65:1
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发表时间：
1999-10
影响因子：
3.8
通讯作者：
E. Shpigel;Arie Goldlust;Gilat Efroni;Amos Avraham;Adi Eshel;M. Dekel;O. Shoseyov
中科院分区：
文献类型：
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作者：
E. Shpigel;Arie Goldlust;Gilat Efroni;Amos Avraham;Adi Eshel;M. Dekel;O. Shoseyov
生物活性蛋白质的固定化对研究和工业具有重要意义。纤维素是一种有吸引力的基质，纤维素结合结构域（CBD）是一种用于纯化和固定许多这些蛋白质的优良亲和标记蛋白。我们构建了两个载体，使融合到CBD的N-或C-末端的蛋白质的克隆和表达。通过将肝素降解蛋白肝素酶I融合到CBD（CBD-HepI和HepI-CBD）来证明它们的有用性。融合蛋白在大肠杆菌中的T7启动子的控制下过表达，并发现在包涵体中积累。通过离心回收包涵体，蛋白质在纤维素柱上重折叠并回收。双功能融合蛋白保留了其结合纤维素和降解肝素的能力。肝素酶I与CBD的C-末端融合在某种程度上上级于N-末端融合：虽然在溶液中的比活性相当，但后者表现出与纤维素的结合能力受损。CBD-HepI-纤维素生物反应器连续运行，并降解肝素超过40小时，而没有任何显着的活性损失。通过改变流速，可以控制所产生的肝素寡糖的平均分子量。分子量分布的配置文件，从肝素解聚游离肝素酶I，游离CBD-HepI，和纤维素固定的CBD-HepI，进行了比较。通过游离肝素酶I和CBD-HepI获得的轮廓是不可区分的，然而，固定化CBD-HepI在相同的解聚百分比下产生低得多的分子量片段。因此，CBD可用于生物反应器的有效生产，将纯化和固定化结合到基本上单一的步骤中。
Immobilization of biologically active proteins is of great importance to research and industry. Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins. We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD. Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD). The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies. The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column. The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin. C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose. CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity. By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled. The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared. The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization. Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step.