Single-stranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering


By jpeza - Posted on 08 Noviembre 2010

Fecha Publicación: 
1 Ene 1986
Nombre de Revista: 
Datos del paper
Autor Principal: 
David A. Mead
Volumen: 
1
Issue: 
1
Página Inicial: 
67
Página Final: 
74
Abstract: 

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 β-galactosidase gene. A
T7
promoter permits in vivo or in vitro
expression of single proteins by the translation of T7 RNA polymerase
transcripts. Insertionsl inactivation of the T7 promoter-containing
β-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 prepropara thyroid
hormone
by oligonucleotide directed mutagenesis. The
mutation changed 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 microsonmi membranes.

Keywords: 
helper phage ; in vitro expression ; membrane translocation ; oligonucleotide mutagenesis ; transcription
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