Heterologous expression and self-assembly of the S-layer protein SbsA of Bacillus stearothermophilus in Escherichia coli.
The cell surface of Bacillus stearothermophilus PV72 is covered by a regular surface layer (S-layer) composed of single species of protein, SbsA, with a molecular weight of 130,000. Recently, the sequence of the corresponding gene (sbsA) has been determined. The SbsA coding region including the signal sequence was cloned as a polymerase chain reaction (PCR) product into a low-copy-number vector under the transcriptional control of the lambda pL promoter. Expression of sbsA was shown to be thermally inducible from the resulting vector pBK4 in a strain of Escherichia coli expressing the lambda cl857 from the chromosome. As shown by ultrathin sectioning of whole cells and immunogold labelling using SbsA- specific antibodies, expression of sbsA in E. coli led to accumulation of sheet-like self-assembling products of the protein in the cytoplasm. No SbsA protein was detected either in the periplasm or in the supernatant fractions. Long-term expression of sbsA from pBK4, including in the late stationary phase, did not lead to degradation of SbsA.
Kuen
B
B
Institut für Mikrobiologie und Genetik, Universität, Austria. oetzi@gem.univie.ac.at
Sára
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M
Lubitz
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D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D005786:Gene Expression Regulation; D001411:Geobacillus stearothermophilus / Q000235:genetics*; D008565:Membrane Proteins; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C088619:sbsA protein, Bacillus stearothermophilus