SINGLE-STRANDED-DNA BLUE-T7 PROMOTER PLASMIDS - A VERSATILE TANDEM PROMOTER SYSTEM FOR CLONING AND PROTEIN ENGINEERING
SINGLE-STRANDED-DNA BLUE-T7 PROMOTER PLASMIDS - A VERSATILE TANDEM PROMOTER SYSTEM FOR CLONING AND PROTEIN ENGINEERING
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DOI：
10.1093/protein/1.1.67
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发表时间：
1986-10-01
期刊：
影响因子：
--
通讯作者：
KEMPER, B
中科院分区：
文献类型：
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作者：
MEAD, DA;SZCZESNASKORUPA, E;KEMPER, B
通过从噬菌体f1克隆单链DNA(ssDNA)复制起点并将噬菌体T7启动子插入β-半乳糖苷酶基因内，从pUC18/19质粒构建嵌合噬菌体质粒表达载体。 T7启动子允许通过T7 RNA聚合酶转录物的翻译在体内或体外表达单一蛋白质。含有T7启动子的β-半乳糖苷酶基因的插入失活允许简单的蓝白色克隆测定。与所检查的几种辅助噬菌体相比，M13K07 的双重感染导致 pTZ 质粒作为 ssDNA 病毒颗粒的最高产量。这些 ssDNA 启动子质粒特别适合蛋白质工程，因为它们简化了克隆、寡核苷酸定向诱变、酶促序列分析验证以及从单个载体表达突变蛋白。这些载体用于通过寡核苷酸定向诱变消除牛前甲状旁腺激素 cDNA 中 T7 RNA 聚合酶的有效转录终止子。该突变将信号肽中的苯丙氨酸 19 密码子更改为丙氨酸。在无细胞系统中，突变的 cDNA 转录本被翻译成前甲状旁腺激素，在微粒体膜存在的情况下，前甲状旁腺激素又转化为甲状旁腺激素原。
Chimeric phage-plasmid expression vectors were constructed from pUC18/19 plasmids by cloning a single-stranded DNA (ssDNA) origin of replication from bacteriophage f1 and inserting a bacteriophage T7 promoter within the .beta.-galactosidase gene. A T7 promoter permits in vivo or in vitro expression of single proteins by the translation of T7 RNA polymerase transcripts. Insertional inactivation of the T7 promoter-containing .beta.-galactosidase gene permits a simple blue-to-white color cloning assay. Compared with several helper phages that were examined, superinfection with M13K07 resulted in the highest yields of the pTZ plasmids as ssDNA viral particles. These ssDNA promoter plasmids are uniquely suited for protein engineering because they simplify cloning, oligonucleotide directed mutagenesis, verification by enzymatic sequence analysis, and expression of mutant proteins from a single vector. These vectors were utilized to eliminate an efficient transcriptional terminator of T7 RNA polymerase in the cDNA of bovine preproparathyroid hormone by oligonucleotide directed mutagenesis. The mutation changes the codon for phenylalanine-19 in the signal peptide to alanine. In a cell-free system the mutant cDNA transcripts were translated into preproparathyroid hormone, which was converted to proparathyroid hormone in the presence of microsomal membranes.