Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis.
Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis.
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通过基因融合和位点特异性蛋白水解快速纯化克隆基因产物。
DOI：
10.1073/pnas.81.15.4692
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发表时间：
1984
影响因子：
11.1
通讯作者：
Bastia,D
中科院分区：
文献类型：
--
作者：
Germino,J;Bastia,D
我们已经开发了一种快速和通用的技术，用于纯化重组DNA克隆中所含顺反子编码的蛋白质。该技术包括通过一段编码接头肽的DNA将正确阅读框中的靶顺反子DNA与标记顺反子融合。此处所示实施例中的靶顺反子是质粒R6 K的复制起始顺反子。接头是编码来自鸡原α-2胶原蛋白的三螺旋区的60个氨基酸的DNA片段，标记顺反子编码大肠杆菌的β-半乳糖苷酶蛋白。通过与β-半乳糖苷酶特异性亲和柱选择性结合并洗脱，快速纯化三重杂合蛋白。然后用纯化的微生物胶原酶消化杂合蛋白以切割接头，并且高压液相色谱允许从标记蛋白快速分离靶蛋白。使用这种技术，我们已经纯化了高度不稳定的R6 K复制起始剂的同质性，我们已经解决了NH 2-末端和COOH-末端片段的蛋白质。通过体外结合，我们进一步表明，COOH末端片段至少有一个DNA结合结构域。该结构域与完整的或β-半乳糖苷酶标记的起始蛋白结合至R6 K染色体的相同限制性片段。
We have developed a rapid and general technique for purification of a protein encoded by a cistron contained in a recombinant DNA clone. The technique consists of fusing the target cistron DNA in the correct reading frame to a marker cistron via a piece of DNA that codes for a linker peptide. The target cistron in the example presented here is the replication initiator cistron of the plasmid R6K. The linker is a DNA fragment encoding 60 amino acids from the triple helical region of chicken pro alpha-2 collagen, and the marker cistron encodes the beta-galactosidase protein of Escherichia coli. The tripartite hybrid protein was rapidly purified by selective binding to and elution from a beta-galactosidase specific-affinity column. The hybrid protein was then digested with a purified microbial collagenase to cleave the linker, and high-pressure liquid chromatography allowed the rapid isolation of the target protein from the marker protein. Using this technique, we have purified the highly labile R6K replication initiator to homogeneity, and we have resolved the protein into NH2-terminal and COOH-terminal segments. We have further shown, by in vitro binding, that the COOH-terminal segment has at least one DNA-binding domain. The domain binds to the same restriction fragments of the R6K chromosome as the intact or beta-galactosidase-tagged initiator protein.