客戶采用我司環(huán)氧基磁珠發(fā)展一種從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
能夠在溫和條件下與粗裂解液中的蛋白質(zhì)共價結(jié)合的功能載體在生物過程中變得越來越重要。本文研制了一種可直接從粗蛋白質(zhì)溶液中共價固定特定蛋白質(zhì)的磁性微球，并對其應(yīng)用條件和穩(wěn)定性進(jìn)行了評價。首先通過遺傳方法用N-末端半胱氨酸修飾SpyCatcher，然后通過環(huán)氧-硫醇反應(yīng)將cys SpyCatcher共價固定在環(huán)氧改性磁珠上。環(huán)氧樹脂與cys SpyCatcher中唯一的末端硫醇基團(tuán)之間的反應(yīng)確保了cys SpyCatcher在顆粒表面的相同取向，而在250mm Na2SO4條件下，cys SpyCatcher的最大結(jié)合容量達(dá)到23.6μg/mg，初始cys SpyCatcher濃度為3 mg/mL時pH值為8.5。功能性磁性微球(MS@cys-SpyCatcher）對與SpyTag（SpyTag-EGFP）融合的增強(qiáng)型綠色熒光蛋白顯示共價位點(diǎn)特異性捕獲特性。（25μg SpyTag-EGFP）/（mg珠）的最大結(jié)合容量遠(yuǎn)遠(yuǎn)高于之前使用SpyCatcher的載體。此外,MS@cys-SpyCatcher可以特異性地直接從細(xì)胞裂解液中捕獲SpyTagged蛋白，并且該載體在pH 4–9和溫度30–70℃下表現(xiàn)出良好的穩(wěn)定性。因此，作為一種通用共價捕獲載體，人們認(rèn)為MS@cys-SpyCatcher可用于生物加工或生物醫(yī)學(xué)應(yīng)用中SpyTagged蛋白質(zhì)的一步純化和定點(diǎn)固定。
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