Gene cloning, functional expression and secretion of the S-layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a in Lactococcus lactis.
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.
Novotny
René
R
Center for NanoBiotechnology, University of Natural Resources and Applied Life Sciences, Wien, Austria.
Scheberl
Andrea
A
Giry-Laterriere
Marc
M
Messner
Paul
P
Schäffer
Christina
C
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003460:Crystallization; D005144:F Factor; D015870:Gene Expression; D005822:Genetic Vectors; D001411:Geobacillus stearothermophilus / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D046912:Multiprotein Complexes / Q000648:ultrastructure; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020539:Sequence Analysis, Protein
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D046912:Multiprotein Complexes; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C055764:S-layer proteins; C490390:SlpB protein, Lactobacillus brevis; C066583:usp45 protein, Lactococcus lactis