Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase.
Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase.
复制标题
开放阅读框表达载体：使用与 β-半乳糖苷酶的蛋白融合在大肠杆菌中产生抗原的通用方法。
DOI：
10.1073/pnas.80.14.4432
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发表时间：
1983
影响因子：
11.1
通讯作者：
Zweig,M
中科院分区：
文献类型：
--
作者：
Weinstock,GM;apRhys,C;Berman,ML;Hampar,B;Jackson,D;Silhavy,TJ;Weisemann,J;Zweig,M
我们已经开发了一种大肠杆菌质粒载体，用于鉴定和表达作为开放阅读框（ORF）的外源DNA片段。ompF的5'端为E.编码丰富的外膜蛋白的大肠杆菌基因，用于提供强的、受调节的启动子、翻译起始位点和信号序列，用于从细胞质输出。该序列与大肠杆菌的lacZ基因偶联。因此β-半乳糖苷酶的表达需要ompF转录和翻译信号。然而，该杂合基因是LacZ-，因为lacZ相对于ompF是框外的。限制酶识别位点位于ompF和lacZ之间，以允许方便地插入DNA片段。如果插入片段是正确长度的ORF，则ompF和lacZ在框中重新对齐，从而产生LacZ+基因，其产生三杂交体蛋白，其中插入片段的翻译产物夹在OmpF和β-半乳糖苷酶之间。因此，LacZ+表型鉴定了含有表达的ORF的克隆。为了证明该载体的效用，我们插入了疱疹病毒胸苷激酶基因的片段，并使用所得的三杂交体蛋白来产生抗体，该抗体从疱疹病毒感染的细胞中沉淀胸苷激酶。我们还插入了E. coli莱克萨基因，以产生三杂交体蛋白，该三杂交体蛋白被用莱克萨蛋白产生的抗血清沉淀。因此，三杂交体融合蛋白可用于产生或检测抗体，也可用于鉴定克隆基因的产物。
We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.