Ubiquitin Fusion System for Recombinant Peptide Expression and Purification : Application to the Cytoplasmic Domain of Syndecan-4
Ubiquitin Fusion System for Recombinant Peptide Expression and Purification : Application to the Cytoplasmic Domain of Syndecan-4
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用于重组肽表达和纯化的泛素融合系统：在 Syndecan-4 细胞质结构域中的应用
DOI：
10.5012/bkcs.2007.28.9.1549
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发表时间：
2007
影响因子：
1.7
通讯作者：
Weontae Lee
中科院分区：
文献类型：
--
作者：
Y. Chae;Hayan Lee;Weontae Lee
syndecan-4，一种I型跨膜硫酸乙酰肝素蛋白聚糖的胞质结构域，在大肠杆菌中过表达为与泛素分子的融合形式，并使用固定化金属亲和层析（IMAC）纯化融合蛋白。通过使用酵母泛素水解酶（YUH）从其融合伴侣释放胞质结构域，随后通过反相色谱纯化。通过MALDI-TOF和NMR光谱检查所得肽片段的完整性。在LB或基本培养基中，肽的产量分别为3.0- 1.5mg/L。该结构域的重组表达和纯化将使我们能够使用多维NMR光谱对其结构和功能进行研究。
The cytoplasmic domain of syndecan-4, a type I transmembrane heparan sulfate proteoglycan, was over-expressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The cytoplasmic domain was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The integrity of the resulting peptide fragment was checked by MALDI-TOF and NMR spectroscopy. The yield of the peptide was 3.0-1.5 mg per liter in LB or minimal medium, respectively. The recombinant expression and purification of this domain will enable us its structural and functional studies using multidimensional NMR spectroscopy.