title: 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.
authors.affiliation: Institute of Genetics, Technische Universität Dresden, Dresden, Germany. Denise.Knobloch@web.de
authors.forename: Denise
authors.initials: D
authors.lastname: Knobloch
authors.forename: Kai
authors.initials: K
authors.lastname: Ostermann
authors.forename: Gerhard
authors.initials: G
authors.lastname: Rödel
mesh_terms: D001410:Bacillus megaterium / Q000235:genetics*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression; D005822:Genetic Vectors; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011523:Protoplasts; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D058644:Sporosarcina / Q000235:genetics*; D014169:Transformation, Bacterial
chemical_list: D003470:Culture Media; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins
references.citation: Curr Microbiol. 2011 Feb;62(2):366-73
references.pmid: 20658344
references.citation: J Bacteriol. 2004 Mar;186(6):1758-68
references.pmid: 14996807
references.citation: Chemphyschem. 2010 Aug 2;11(11):2323-6
references.pmid: 20629069
references.citation: Anal Biochem. 1984 Apr;138(1):141-3
references.pmid: 6731838
references.citation: Microb Cell Fact. 2005 Oct 04;4:28
references.pmid: 16202167
references.citation: Mol Microbiol. 1996 Sep;21(6):1117-23
references.pmid: 8898381
references.citation: Appl Environ Microbiol. 2009 Jun;75(12):3891-5
references.pmid: 19395560
references.citation: J Biol Chem. 1991 Aug 15;266(23):15258-65
references.pmid: 1869553
references.citation: Mol Microbiol. 1997 Oct;26(2):277-88
references.pmid: 9383153
references.citation: Bioconjug Chem. 2009 May 20;20(5):895-903
references.pmid: 19402706
references.citation: Langmuir. 2011 Apr 5;27(7):3731-8
references.pmid: 21401053
references.citation: Microb Cell Fact. 2011 Feb 10;10:6
references.pmid: 21310062
references.citation: Appl Environ Microbiol. 2002 Dec;68(12):5943-51
references.pmid: 12450814
references.citation: J Bacteriol. 1992 Nov;174(22):7419-27
references.pmid: 1429463
references.citation: Microbiology (Reading). 1997 Apr;143 ( Pt 4):1039-1052
references.pmid: 9141671
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: J Bacteriol. 1998 Mar;180(6):1488-95
references.pmid: 9515918
references.citation: Appl Environ Microbiol. 2007 May;73(10):3245-53
references.pmid: 17384306
references.citation: J Bacteriol. 1997 Jun;179(12):3892-8
references.pmid: 9190804
references.citation: Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W197-201
references.pmid: 18463136
references.citation: Appl Environ Microbiol. 2002 Jul;68(7):3251-60
references.pmid: 12089001
references.citation: Biochem J. 2004 Apr 15;379(Pt 2):441-8
references.pmid: 14725506
references.citation: Microbiology (Reading). 2001 May;147(Pt 5):1353-1363
references.pmid: 11320138
references.citation: J Struct Biol. 1998 Dec 15;124(2-3):276-302
references.pmid: 10049812
references.citation: J Bacteriol. 1989 Dec;171(12):6747-52
references.pmid: 2512285
references.citation: FEMS Microbiol Lett. 2002 Dec 17;217(2):263-7
references.pmid: 12480114
references.citation: FEMS Microbiol Lett. 2005 Jan 1;242(1):27-35
references.pmid: 15675069
references.citation: Bioconjug Chem. 2008 Apr;19(4):860-5
references.pmid: 18376854
references.citation: Protein Eng. 2002 Mar;15(3):243-9
references.pmid: 11932495
references.citation: Annu Rev Microbiol. 1983;37:311-39
references.pmid: 6416145
references.citation: Appl Microbiol Biotechnol. 2007 Oct;76(5):957-67
references.pmid: 17657486
references.citation: Bioconjug Chem. 2003 Mar-Apr;14(2):440-8
references.pmid: 12643755
references.citation: J Bacteriol. 1987 Mar;169(3):1239-45
references.pmid: 3029027
references.citation: Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):14646-51
references.pmid: 12417763
references.citation: J Nanosci Nanotechnol. 2005 Dec;5(12):1939-53
references.pmid: 16430129
references.citation: FEMS Microbiol Rev. 1997 Jun;20(1-2):47-98
references.pmid: 9276928
references.citation: J Gen Microbiol. 1957 Aug;17(1):227-37
references.pmid: 13475687
references.citation: J Nanosci Nanotechnol. 2011 Jan;11(1):402-7
references.pmid: 21446464
references.citation: Appl Microbiol Biotechnol. 1995 Mar;42(6):871-7
references.pmid: 7766087
references.citation: J Bacteriol. 2000 Feb;182(4):859-68
references.pmid: 10648507
references.citation: Nucleic Acids Res. 2006 Jul 1;34(Web Server issue):W362-5
references.pmid: 16845026
references.citation: Microbiology (Reading). 2005 Mar;151(Pt 3):643-651
references.pmid: 15758211
references.citation: Microbiology (Reading). 2000 Feb;146 ( Pt 2):273-281
references.pmid: 10708365