Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation.
An inactivated gene for Bacillus amyloliquefaciens extracellular ribonuclease (barnase) has previously been cloned and sequenced following transposon mutagenesis. The intact gene could not be assembled in Escherichia coli and is presumed to be lethal. Therefore, we introduced specific mutations into the barnase gene to prevent its lethal effect. A Gln-73 mutant gene was stable in E. coli but only produced low amounts of barnase antigen. Mutants containing Asp, Gln or Arg, instead of His-102, at the active site were identified by immunological screening for barnase antigen. None of the mutant proteins with alterations at aa residue 102 possessed RNase activity. The level of barnase (Asp-102) was higher in E. coli than in B. subtilis but the protein was not processed to the correct size in E. coli. To obtain correct processing, the barnase (Asp-102) structural gene was fused to the E. coli alkaline phosphatase promoter and signal sequence (phoA). Cells containing this construct secreted correctly processed barnase (Asp-102) into the periplasmic space and culture supernatant at a level of 20 mg/l. Barnase (Asp-102) was purified and found to have an identical N-terminus and a thermal unfolding curve that was nearly identical to that of active barnase (His-102). The cloning and expression of barnase in E. coli will allow detailed analysis of barnase protein folding by molecular genetic approaches.
Paddon
C J
CJ
Hartley
R W
RW
D000469:Alkaline Phosphatase / Q000235:genetics; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D007158:Immunologic Techniques; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D012260:Ribonucleases / Q000235:genetics* / Q000276:immunology
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D000469:Alkaline Phosphatase