Construction of an efficient overproducer clone of HinfI restriction endonuclease using the polymerase chain reaction.
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.
Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205.
C M
CM
Skoglund
H O
HO
Smith
S
S
Chandrasegaran
D001483:Base Sequence; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005809:Genes, Regulator; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction
D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases