Gene cloning, functional expression and secretion of the S-layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a in Lactococcus lactis |
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Authors: | Novotny René Scheberl Andrea Giry-Laterriere Marc Messner Paul Schäffer Christina |
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Affiliation: | Center for NanoBiotechnology, University of Natural Resources and Applied Life Sciences, Wien, Austria. |
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Abstract: | The ~93-kDa surface layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a forms a regular crystalline array providing a nanopatterned matrix for the future display of biologically relevant molecules. Lactococcus lactis NZ9000 was established as a safe expression host for the controlled targeted production of SgsE based on the broad host-range plasmid pNZ124Sph, into which the nisA promoter was introduced. SgsE devoid of its signal peptide-encoding sequence was cloned into the new vector and purified from the cytoplasm at a yield of 220 mg l- of expression culture. Secretion constructs were based on the signal peptide of the Lactobacillus brevis SlpA protein or the L. lactis Usp45 protein, allowing isolation of 95 mg of secreted rSgsE l-1. N-terminal sequencing confirmed correct processing of SgsE in L. lactis NZ9000. The ability of rSgsE to self-assemble in suspension and to recrystallize on solid supports was demonstrated by electron and atomic force microscopy. |
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Keywords: | S-layer protein Geobacillus stearothermophilus Functional expression Secretion Lactococcus lactis |
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