客户采用我司环氧基磁珠发展一种从粗蛋白中直接定向固定化蛋白的方法
Mianxing Luo, et al. A potential method for one-step purification and direct immobilization of target protein in cell lysate with magnetic microbeads,Biochemical Engineering Journal, Volume 176, December 2021, 108182
Abstract
Functional carrier capable of covalently binding proteins from crude lysate under mild condition has become increasingly important in bioprocess. In this paper, a magnetic microbead that can covalently immobilize specific proteins directly from crude protein solution was developed, and its application conditions and stability were evaluated. The SpyCatcher was firstly modified with N-terminal cysteine by genetic methods and then the cys-SpyCatcher was covalently immobilized on epoxy-modified magnetic beads by epoxy-thiol reaction. The reaction between the epoxy and the only terminal thiol group in cys-SpyCatcher ensured the same orientation of the cys-SpyCatcher on the particle surface, while the maximum binding capacity of cys-SpyCatcher reached 23.6 μg/mg under the condition of 250 mM Na2SO4, pH 8.5 at initial cys-SpyCatcher concentration of 3 mg/mL. The functional magnetic microsphere (MS@cys-SpyCatcher) showed covalent site-specific capture property for enhanced green fluorescent protein fused with SpyTag (SpyTag-EGFP). A maximum binding capacity of (25 μg SpyTag-EGFP)/(mg beads) was far more than that of the previous carrier with SpyCatcher. In addition, the MS@cys-SpyCatcher could specifically capture the SpyTagged protein directly from the cell lysate, and this carrier showed good stability at pH 4–9 and temperature of 30–70ºC. Therefore, as a universal covalent capture carrier, it is believed that the MS@cys-SpyCatcher is useful in one-step purification and site-specific immobilization of the SpyTagged protein in bioprocess or biomedical application.
摘要
能够在温和条件下与粗裂解液中的蛋白质共价结合的功能载体在生物过程中变得越来越重要。本文研制了一种可直接从粗蛋白质溶液中共价固定特定蛋白质的磁性微球，并对其应用条件和稳定性进行了评价。首先通过遗传方法用N-末端半胱氨酸修饰SpyCatcher，然后通过环氧-硫醇反应将cys SpyCatcher共价固定在环氧改性磁珠上。环氧树脂与cys SpyCatcher中唯一的末端硫醇基团之间的反应确保了cys SpyCatcher在颗粒表面的相同取向，而在250mm Na2SO4条件下，cys SpyCatcher的最大结合容量达到23.6μg/mg，初始cys SpyCatcher浓度为3 mg/mL时pH值为8.5。功能性磁性微球(MS@cys-SpyCatcher）对与SpyTag（SpyTag-EGFP）融合的增强型绿色荧光蛋白显示共价位点特异性捕获特性。（25μg SpyTag-EGFP）/（mg珠）的最大结合容量远远高于之前使用SpyCatcher的载体。此外,MS@cys-SpyCatcher可以特异性地直接从细胞裂解液中捕获SpyTagged蛋白，并且该载体在pH 4–9和温度30–70℃下表现出良好的稳定性。因此，作为一种通用共价捕获载体，人们认为MS@cys-SpyCatcher可用于生物加工或生物医学应用中SpyTagged蛋白质的一步纯化和定点固定。
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