Department of Bioengineering and Biotechnology, Huaqiao University, Jimei Ave. 668, Xiamen 361021, China.
Department of Bioengineering and Biotechnology, Huaqiao University, Jimei Ave. 668, Xiamen 361021, China.
Int J Biol Macromol. 2024 Nov;280(Pt 4):135811. doi: 10.1016/j.ijbiomac.2024.135811. Epub 2024 Sep 24.
Owing to the rapid advancement of in vitro synthetic biology, functional carriers capable of covalently binding target proteins from crude lysates under mild conditions have garnered escalating attention. Herein, a magnetic nanoparticle with affinity/covalent bifunction (MNP@Affi-Cova) was developed for the direct covalent immobilization of the recombinant enzyme of His-tagged birA (r-birA) from crude cell lysates in a single step. This innovative approach is attributed to the presence of chelated Ni ions and epoxy groups on the surface of the beads. The fabricated magnetic nanoparticles were characterized by SEM, FT-IR spectrum, and zeta potential. The application conditions and stability of the MNP@Affi-Cova beads were systematically evaluated. Notably, the MNP@Affi-Cova beads exhibited a covalent capture efficiency of 91.25 μg r-birA/mg beads from a cell lysate supernatant containing 2.62 mg/mL crude protein. The immobilized r-birA exhibited significantly enhanced pH and thermal stability compared to the free counterpart. Additionally, the reusability of the immobilized r-birA on MNP@Affi-Cova demonstrated the retention of 76.1 % of its initial activity over ten cycles. These results suggest that the MNP@Affi-Cova presents considerable potential as a support for the covalent immobilization of recombinant His-tagged enzymes directly from crude lysates, thereby circumventing the labor-intensive purification process typically required before enzyme immobilization.
由于体外合成生物学的快速发展，能够在温和条件下共价结合粗裂解物中靶蛋白的功能载体引起了越来越多的关注。本文开发了一种具有亲和/共价双功能的磁性纳米颗粒（MNP@Affi-Cova），可用于一步直接共价固定来自粗细胞裂解物的 His 标记的 birA 的重组酶（r-birA）。这种创新方法归因于珠子表面螯合的 Ni 离子和环氧基团的存在。通过 SEM、FT-IR 光谱和zeta 电位对制备的磁性纳米颗粒进行了表征。系统评估了 MNP@Affi-Cova 珠的应用条件和稳定性。值得注意的是，从含有 2.62mg/mL 粗蛋白的细胞裂解物上清液中，MNP@Affi-Cova 珠对 91.25μg r-birA/mg 珠的共价捕获效率达到 91.25μg r-birA/mg 珠。固定化 r-birA 的 pH 值和热稳定性明显优于游离对照物。此外，MNP@Affi-Cova 上固定化 r-birA 的可重复使用性表明，在十个循环中保留了其初始活性的 76.1%。这些结果表明，MNP@Affi-Cova 具有相当大的潜力，可作为直接从粗裂解物共价固定重组 His 标记酶的载体，从而避免了酶固定化前通常需要的劳动密集型纯化过程。
