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Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium.

ABSTRACT: Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

SUBMITTER: Knobloch D

PROVIDER: S-EPMC3255725 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium.

Knobloch Denise D Ostermann Kai K Rödel Gerhard G

Applied and environmental microbiology 20111118 2

Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-t ...[more]

PMID: 22101038

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Project description:A recombinant B. megaterium strain was used for the heterologous production of a glucosyltransferase (dextransucrase). To better understand the physiological and metabolic responses of the host cell to cultivation and induction conditions, proteomic analysis was carried out by combined use of two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) for protein separation and identification. 2-DE method was optimized for the separation of intracellular proteins. Since the genome of B. megaterium is not yet available, peptide sequencing using peptide fragment information obtained from nanoelectrospray ionization quadrupole-time-of-flight tandem mass spectrometry (ESI-QqTOF MS/MS) was applied for protein identification. 167 protein spots were identified as 149 individual proteins, including most enzymes involved in the central carbon metabolic pathways and many enzymes related to amino acid synthesis and protein synthesis. Based on the results a 2-DE reference map and a corresponding protein database were constructed for further proteomic approaches on B. megaterium. For the first time it became possible to perform comparative proteomic analysis on B. megaterium in a batch culture grown on glucose with xylose induction for dextrasucrase production. No significant differences were observed in the expression changes of enzymes of the glycolysis and TCA cycle, indicating that dextransucrase production, which amounted to only 2 % of the entire protein production, did not impose notable metabolic or energetic burdens on the central carbon metabolic pathway of the cells. However, a short-term up-regulation of aspartate aminotransferase, an enzyme closely related to dextransucrase production, in the induced culture demonstrated the feasibility to use 2-DE method for monitoring dextransucrase production. It was also observed that under the cultivation conditions used in this study B. megaterium tended to channel acetyl-CoA into pathways of polyhydroxybutyrate production. No expression increases were found with cytosolic chaperones such as GroEL and DnaK during dextransucrase production and secretion, whereas a strong up-regulation of the oligopeptide-binding protein OppA was observed in correlation with an increased secretion of dextransucrase into the culture medium.

Dataset Information

Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium.

Publications

Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium.

Similar Datasets

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