Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

A cDNA fragment encoding the S-layer protein SllB cloned from Bacillus sphaericus ATCC 14577 was expressed on the surface of E. coli BL21 (DE3) cells and confirmed by the square lattice structure at the nanoscale level. The amplified gene fragment designed with PCR primers from a specified reference sequence (GenBank accession no. AJ849550) showed a high degree of sequence identity with the known sequences for S-layer protein. The best alignment scores were seen in B. sphaericus strains JG-A12 and NCTC9602, which code for a pre-form protein with a predicted cleavage site located between the two alanine residues 31 and 32. After this signal peptide sequence was removed, the mature protein had a molecular mass of 116.2613 kDa and a theoretical pI of 5.40. Further bioinformatic analysis revealed three S-layer homology (SLH) domains in the N-terminus of the mature protein, positioned at the 1–61, 63–128 and 137–197 residues. The mature S-layer protein was composed of alpha helices (24.86%), extended strands (27.01%), and rich random coils (48.13%). Bioinformatics-driven characterization of SllB may provide scientific evidence for further application of this gene in the fields of nanobiotechnology and biomimetics in the future.

[1]  Yubao Cui,et al.  Cloning, Expression, and Characterization of Der f 7, an Allergen of Dermatophagoides farinae from China , 2010, Journal of medical entomology.

[2]  Xiaomin Hu,et al.  Species-Specific Cell Wall Binding Affinity of the S-Layer Proteins of Mosquitocidal Bacterium Bacillus sphaericus C3-41 , 2009, Applied and Environmental Microbiology.

[3]  T. Baldet,et al.  Impact of Slow-Release Bacillus Sphaericus Granules on Mosquito Populations Followed in a Tropical Urban Environment , 2009, Journal of medical entomology.

[4]  K. Zarschler,et al.  S-layer nanoglycobiology of bacteria. , 2008, Carbohydrate research.

[5]  D. Pum,et al.  S-layers as a tool kit for nanobiotechnological applications. , 2007, FEMS microbiology letters.

[6]  D. Pum,et al.  S‐Layers as a basic building block in a molecular construction kit , 2007, The FEBS journal.

[7]  L. Lacey.  BACILLUS THURINGIENSIS SEROVARIETY ISRAELENSIS AND BACILLUS SPHAERICUS FOR MOSQUITO CONTROL , 2007, Journal of the American Mosquito Control Association.

[8]  G. Rödel,et al.  Isolation, gene structure, and comparative analysis of the S-layer gene sslA of Sporosarcina ureae ATCC 13881 , 2007, Genetica.

[9]  D. Pum,et al.  S-layer proteins as key components of a versatile molecular construction kit for biomedical nanotechnology. , 2006, Mini reviews in medicinal chemistry.

[10]  M. El-Bendary,et al.  Bacillus thuringiensis and Bacillus sphaericus biopesticides production , 2006, Journal of basic microbiology.

[11]  Katrin Pollmann,et al.  Novel surface layer protein genes in Bacillus sphaericus associated with unusual insertion elements. , 2005, Microbiology.

[12]  J. Harris,et al.  Molecular organization of selected prokaryotic S-layer proteins. , 2005, Canadian journal of microbiology.

[13]  P. Messner,et al.  The structure of secondary cell wall polymers: how Gram-positive bacteria stick their cell walls together. , 2005, Microbiology.

[14]  D. Pum,et al.  Functionalisation of surfaces with S-layers. , 2004, IEE proceedings. Nanobiotechnology.

[15]  U. Sleytr,et al.  Interaction of the Crystalline Bacterial Cell Surface Layer Protein SbsB and the Secondary Cell Wall Polymer of Geobacillus stearothermophilus PV72 Assessed by Real-Time Surface Plasmon Resonance Biosensor Technology , 2004, Journal of bacteriology.

[16]  Mittal Pk.  Biolarvicides in vector control: challenges and prospects. , 2003 .

[17]  P. K. Mittal.  Biolarvicides in vector control: challenges and prospects. , 2003, Journal of vector borne diseases.

[18]  Dietmar Pum,et al.  S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Andreas Breitwieser,et al.  A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen. , 2002, Protein engineering.

[20]  M. Mock,et al.  Bacterial SLH domain proteins are non‐covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation , 2000, The EMBO journal.

[21]  I. Olsen,et al.  S-layers of Bacillus species. , 1997, Microbiology.