Construction of an efficient overproducer clone of HinfI restriction endonuclease using the polymerase chain reaction

Cynthia M. Skoglund, Hamilton O. Smith, Srinivasan Chandrasegaran

Research output: Contribution to journalArticlepeer-review

Abstract

We describe the use of the polymerase chain reaction (PCR) technique to alter transcriptional and translational signals surrounding a gene so as to achieve overexpression in Escherichia coli. By changing the ribosome-binding site sequence preceding the hinfIR gene to match the consensus E. coli signal and by adding a transcription terminator sequence immediately following the gene, the yield of HinfI was increased about tenfold over that obtained from the natural Haemophilus influenzae signals. The addition of the positive retroregulator stem-loop sequence derived from the crystal protein-encoding gene of Bacillus thuringiensis downstream from the hinfIR gene further increased yields by twofold to a level of 13% of the total cellular protein.

Original languageEnglish (US)
Pages (from-to)1-5
Number of pages5
JournalGene
Volume88
Issue number1
DOIs
StatePublished - Mar 30 1990

Keywords

  • Escherichia coli
  • Recombinant DNA
  • overexpression
  • plasmid
  • positive retroregulator
  • ribosome-binding site
  • stem-loop structure

ASJC Scopus subject areas

  • Genetics

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