Production of low-expressing recombinant cationic biopolymers with high purity.
Production of low-expressing recombinant cationic biopolymers with high purity.
复制标题
DOI：
10.1016/j.pep.2017.03.012
复制
发表时间：
2017-06
影响因子：
1.6
通讯作者：
Hatefi A
中科院分区：
文献类型：
--
作者：
Chen X;Nomani A;Patel N;Hatefi A
用于输送生物活性物质的重组生物聚合物日益复杂，这就要求能够高精度地控制生物材料的结构。基因工程技术提供了在生物体中合成生物材料的机会，例如完全控制其长度和序列的大肠杆菌。一类这样的生物聚合物是重组阳离子生物聚合物，应用于基因传递、再生医学和各种其他生物医学应用。遗憾的是，由于它们的阳离子性质和复杂的结构，它们在大肠杆菌表达系统中的表达效率很低，这使得获得纯生物聚合物的可能性变得更加复杂。SlyD和Arna内源性大肠杆菌蛋白被认为是在金属亲和层析过程中与低表达生物聚合物相互作用的主要罪魁祸首。在这里，我们比较了不同的参数，如表达宿主的选择以及金属亲和柱的影响，以确定最有效的方法来获得高纯度和可接受的产量的重组阳离子生物聚合物。研究结果表明，利用大肠杆菌BL21(DE3)LobSTR菌株，结合我们开发的严格表达和Ni-NTA纯化方案，一步纯化即可获得高纯度的产物(纯度为99%)。该方法可用于生产其他具有广泛生物医学应用的复杂和潜在有毒的生物聚合物。
The growing complexity of recombinant biopolymers for delivery of bioactive agents requires the ability to control the biomaterial structure with high degree of precision. Genetic engineering techniques have provided this opportunity to synthesize biomaterials in an organism such as E. coli with full control over their lengths and sequences. One class of such biopolymers is recombinant cationic biopolymers with applications in gene delivery, regenerative medicine and variety of other biomedical applications. Unfortunately, due to their highly cationic nature and complex structure, their production in E. coli expression system is marred by low expression yield which in turn complicates the possibility of obtaining pure biopolymer. SlyD and ArnA endogenous E. coli proteins are considered the major culprits that copurify with the low-expressing biopolymers during the metal affinity chromatography. Here, we compared the impact of different parameters such as the choice of expression hosts as well as metal affinity columns in order to identify the most effective approach in obtaining highly pure recombinant cationic biopolymers with acceptable yield. The results of this study showed that by using E. coli BL21(DE3) LOBSTR strain and in combination with our developed stringent expression and Ni-NTA purification protocols highly pure products in one purification step (>99% purity) can be obtained. This approach could be applied to the production of other complex and potentially toxic biopolymers with wide range of applications in biomedicine.