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

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 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 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 microsomal membranes.

Isolation and Characterization of the cDNA Encoding Bovine Poly(ADP-ribose) Glycohydrolase

“…Clones 6 -8 provided overlapping sequences for the 5Ј-terminal region. The full-length cDNA was constructed by ligating a 3.9-kb XbaI-NsiI fragment from pWL11 (clone 1 cDNA insert in pTZ18R (36)) and a 3.0-kb NsiI-XbaI fragment from pWL13 (clone 4 cDNA insert in pTZ18R). The resulting plasmid, termed pWL30, contained the 4,070-bp full-length cDNA.…”

Inositol monophosphatase activity from the Escherichia coli suhB gene product

“…The KpnI site and a ribosome binding sequence on the 5Ј side and an EcoRI site on the 3Ј side were introduced with these primers. The PCR product was cloned into the KpnI and EcoRI sites of pTZ19R (23), producing pKS262. The unique EcoRI site of this plasmid was converted to a BamHI site by ligating a BamHI linker (CGGATCCG) to the EcoRI site that was made flush by treatment with a Klenow fragment, producing pKS277.…”

Structure‐function relationship in the globular type III antifreeze protein: Identification of a cluster of surface residues required for binding to ice

“…The mutagenic oligonucleotides used are listed in Figure 1. In addition to the use of plasmid pTZ-19r (Mead et al, 1986) for the generation of single-stranded, uracil-containing template, a second plasmid pT7-7(f) was also used. pT7-7(f) was made by the incorporation of an fl origin of replication into the expression vector pT7-7 (Tabor, 1990) as previously described (DeLuca et al, 1993).…”