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首页> 美国政府科技报告 >Single-Stranded DNA 'Blue' T7 Promoter Plasmids: A Versatile Tandem Promoter System for Cloning and Protein Engineering
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Single-Stranded DNA 'Blue' T7 Promoter Plasmids: A Versatile Tandem Promoter System for Cloning and Protein Engineering

机译：单链DNa'Blue'T7启动子质粒：用于克隆和蛋白质工程的多功能串联启动子系统

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摘要

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 transcripts were translated into preproparathyroid hormone, which was converted to proparathyroid hormone in the presence of microsomal membranes.

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