Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E. coli.
A new phagemid vector, pSIT, was constructed that allows both oligonucleotide-directed mutagenesis and tightly controlled, high-level expression of proteins in Escherichia coli. An efficient rate of mutagenesis is achieved by taking advantage of the double oligonucleotide primer technique. In addition to the mutagenic primer, a second oligonucleotide primer conferring antibiotic resistance to the mutant DNA strand is annealed to single-strand DNA. Selection for the antibiotic thus increases the frequency of mutants. High-level and tightly controlled expression of heterologous proteins is enabled by utilizing a very strong hybrid T7lac promoter and lac repressor in conjunction with T7 RNA polymerase, as well as a high copy number of the vector. The pSIT phagemid permits overexpression of proteins and their mutants without subclonings from mutagenic to expression constructs, which saves time, especially when multiple mutations of the same protein are proposed. A retroviral proteinase precursor, toxic for E. coli, was successfully expressed to a high level, and a series of mutants of this protein was readily obtained.
University of Alabama at Birmingham.
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M
Andreansky
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E
Hunter
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D010450:Endopeptidases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D016093:Mason-Pfizer monkey virus / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics; D015183:Restriction Mapping
D011498:Protein Precursors; D004262:DNA Restriction Enzymes; D010450:Endopeptidases