BETA-GLUCURONIDASE FROM ESCHERICHIA-COLI AS A GENE-FUSION MARKER
BETA-GLUCURONIDASE FROM ESCHERICHIA-COLI AS A GENE-FUSION MARKER
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DOI：
10.1073/pnas.83.22.8447
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发表时间：
1986-11-01
影响因子：
11.1
通讯作者：
HIRSH, D
中科院分区：
文献类型：
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作者：
JEFFERSON, RA;BURGESS, SM;HIRSH, D
我们已经开发了一种基于大肠杆菌β-葡萄糖醛酸酶基因（uidA）。uidA基因已从大肠杆菌中克隆。coliK-12中表达，并测定了其核苷酸序列。β-葡糖醛酸酶已纯化至均一并进行了表征。该酶具有68，200的亚基分子量，非常稳定，并且使用市售底物容易且灵敏地测定。我们构建了E. coli lacZ启动子和编码区与uidA基因的编码区，其显示β-乳糖控制下的葡萄糖醛酸酶活性。已经构建了质粒载体以促进β-通过使用许多不同的限制性内切酶切割位点，将葡糖醛酸酶编码区与异源控制区连接。存在几种生物系统，其中uidA编码的β-葡糖醛酸糖苷酶可以是先前描述的基因融合标记物如β-葡糖苷酸酶的有吸引力的替代物或补充物。半乳糖苷酶或氯霉素乙酰转移酶。
We have developed a gene-fusion system based on the Escherichia coli .beta.-glucuronidase gene (uidA). The uidA gene has been cloned from E. coli K-12 and its entire nucleotide sequence has been determined. .beta.-Glucuronidase has been purified to homogeneity and characterized. The enzyme has a subunit molecular weight of 68,200, is very stable, and is easily and sensitively assayed using commercially available substrates. We have constructed gene fusions of the E. coli lacZ promoter and coding region with the coding region of the uidA gene that show .beta.-glucuronidase activity under lac control. Plasmid vectors have been constructed to facilitate the transfer of the .beta.-glucuronidase coding region to heterologous control regions, using many different restriction endonuclease cleavage sites. There are several biological systems in which uidA-encoded .beta.-glucuronidase may be an attractive alternative or complement to previously described gene-fusion markers such as .beta.-galactosidase or chloramphenicol acetyltransferase.