Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E. coli

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
JournalBioTechniques
Volume16
Issue number4
StatePublished - Jan 1 1994
Externally publishedYes

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Mutagenesis
DNA Primers
Mutant Proteins
Escherichia coli
Lac Repressors
Poisons
Escherichia coli Proteins
DNA
Microbial Drug Resistance
Site-Directed Mutagenesis
Anti-Bacterial Agents
Proteins
Peptide Hydrolases
Oligonucleotides
Mutation
In Vitro Techniques
bacteriophage T7 RNA polymerase

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Clinical Biochemistry

Cite this

Phagemid pSIT permits efficient in vitro mutagenesis and tightly controlled expression in E. coli. / Andreansky, Martin; Hunter, E.

In: BioTechniques, Vol. 16, No. 4, 01.01.1994.

Research output: Contribution to journalArticle

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