Synthesis of the Bacillus subtilis histone-like DNA-binding protein HBsu in Escherichia coli and secretion into the periplasm.
A synthetic gene encoding the histone-like DNA-binding protein, HBsu, of Bacillus subtilis was cloned in-frame behind the coding region of the OmpA signal peptide of Escherichia coli. The gene encoding the fusion protein is under control of both the lpp promoter and the lac promoter-operator. Upon induction of gene expression, mature HBsu is secreted into the periplasm. The OmpA signal peptide is correctly removed, resulting in the production of authentic-length HBsu protein. The observed in vitro DNA-binding ability is taken as evidence for the correct folding and assembly of homodimeric HBsu protein. A normally intracellular protein can thus be secreted from E. coli in high yield and with full functionality. By analogy, every histone-like protein or mutant forms thereof may be produced heterologously in E. coli and may be purified without being contaminated by the homologous E. coli HU protein.
Groch
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N
Freie Universität Berlin, Institut für Kristallographie, FRG.
Hahn
U
U
Heinemann
U
U
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D006657:Histones; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C075779:Hbsu protein, bacteria; D006657:Histones; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins