High level heterologous expression in E. coli using mutant forms of the lac promoter.
Single base deletions in the lac promoter which reduced the 18bp spacing between the -35 and -10 homology regions to 17bp, increased the strength of the promoter. A single base substitution (T----G) in the -35 region to generate the consensus sequence TTG-ACA increased the strength further and no longer required a 17bp spacing. The mutated lac promoter was as powerful as a shorter form of the tac promoter which lacked two AT-rich regions upstream of the -35 region, and expressed the P69 surface antigen (pertactin) of Bordetella pertussis to 30-40% total cell protein and tetanus toxin fragment C to 16-20% total cell protein.
Makoff
A J
AJ
Wellcome Research Laboratories, Beckenham, Kent, UK.
Oxer
M D
MD
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D015152:Blotting, Northern; D001886:Bordetella pertussis / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D010566:Virulence Factors, Bordetella
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D012333:RNA, Messenger; D010566:Virulence Factors, Bordetella; C068929:pertactin