ATCC 14577 was provided by the Agricultural Culture Collection of China (ACCC). It is routinely grown in nutrient broth (NB) medium consisting of 5 g peptone 1⁻¹ and 3 g meat extract 1⁻¹. pMD19-T Simple Vector (code no. D104, Takara Biotechnology Limited Company, Dalian, China) and pET28a (+) (kit lot no. N72770 Novagen, Germany) were used for the cloning and expression, respectively. Escherichia coli JM109 competent cells (code no. D9052, Takara) used in cloning; and E. coli BL21 used in expression (DE3, Stratagene, USA), were cultivated at 37°C on agar plates and in broth medium.

Genomic DNA of B. sphaericus ATCC 14577 was prepared, with the MiniBEST Bacterial Genomic DNA Extraction kit Ver.2.0 (code no. DV810, Takara) according to the manufacturer's instructions. According to the published sequence (GenBank accession no. AJ849550), the oligonucleotide primers were designed with specific sequences, 5′-GGATCCATGGCTAACCAACCAA AGAAATAC-3′ (forward) and 5′-CTCGAGTTATGGAG TAGGCTTTACTGTAATAG-3′ (reverse). These contained a BamHI site and a XhoI site at their 5′ ends (underlined), respectively. Using genomic DNA from B. sphaericus ATCC 14577 as the template, the sllB gene encoding S-layer was amplified by PCR. The reaction system was prepared with the PrimeSTAR^®HS DNA Polymerase with GC Buffer (code no. DR044A, Takara), and the total reaction mixture contained 1 μl of genomic DNA, 25 μl of 2X PrimeSTAR GC (Mg₂+plus) Buffer, 4 μl of dNTP mixture (25 mM each), 1 μl each of the forward and reverse primer (20 μM each), 0.5 μl of PrimeSTAR HS DNA Polymerase (2.5 U/μl) and 17.5 μl of dH₂O. PCR conditions included an initial incubation at 94°C for 3 min, followed by 30 cycles at 98°C for 10 sec, at 55°C for 15 sec and at 72°C for 3 min. After a final incubation at 72°C for 10 min, 5 μl of the amplicons were analyzed by agarose gel electrophoresis (1.0%) and visualized with ImageMaster^® VDS.

The PCR-amplified DNA was recovered from the gel with Agarose Gel DNA Purification kit ver. 2.0 (code no. DV805, Takara), and then a poly-A tail was added with DNA A-Tailing kit (code no. D404, Takara). The poly-A tailed product was then cloned into the simple vector pMD19-T (code no. D104, Takara). Then, E. coli JM109 (code no. D9052, Takara) was transformed with the recombinant plasmid pMD19-T-sllB and positive clones selected by blue/white screening on Luria-Bertani (LB) plates. The inserts were confirmed by restriction enzyme analysis using BamHI and XhoI and then sequenced using Primer pMD18F, Primer pMD18R, seqF1 (5′-TTCGCTTCATTCTTATA CCG-3′), seqF2 (5′-ACTTACAAACACCCAGAAAC-3′), seqF3 (5′-CAAAGCGGTAAAGATGCAA-3′) in an ABI PRISM™ 377XL DNA Sequencer (Takara). After alignment of the sequenced results and the reference sequence, a recombinant pMD19-T-sllB plasmid with the most homologous reference sequence was digested with BamHI and XhoI. To release the sllB fragment, the Agarose Gel DNA Purification kit ver. 2.0 (code no. DV805, Takara) was used. Utilization of the DNA Ligation kit (code no. D6023, Takara) allowed creation of pET28a(+)-sllB via sub-cloning of the sllB cDNA fragment into the expression vector pET28a(+). Competent E. coli JM109 cells were transformed with pET28a(+)-sllB plasmids, positive clones were selected by blue/white screening, and then confirmed by restriction enzyme analysis with BamHI and XhoI.

A pET28a(+)-sllB plasmid was prepared using the MiniBEST Plasmid Purification kit ver. 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene). The E. coliBL21 cells carrying pET28a (+)-sllB were grown overnight at 37°C on LB plates containing 50 μg/ml of kanamycin. A single colony was inoculated into 5 ml LB containing 50 μg/ml of kanamycin and then cultured at 37°C. Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added (50 μl of a 100 mM stock, final concentration 1 mM) for 3 h to induce the tac promoter. The E. coli cells were pelleted by centrifugation, resuspended in PBS buffer (200 μl/tube), and ultrasonicly disrupted. One aliquot (50 μl) was harvested to represent whole cell lysate. The remaining lysate was centrifuged to separate the supernatant and pellet, which contained soluble and insoluble proteins, respectively.

The whole cell lysate, soluble protein fraction, and insoluble protein fraction (10 μl each) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12.5% polyacrylamide gel), followed by CBB-R250 staining. After electrophoresis, the proteins were transferred (39 mA, 80 min) to a PVDF membrane (Tiangen Biotech Co. Led, Beijing, China) and incubated with blocking buffer (1.5% of BSA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.1% Tween-20). The membrane was then incubated at 4°C overnight with 1:1000 primary Penta-His antibody (Qiagen, Germany) primary antibody, followed by incubation at room temperature for 1 h with 1:1000 horseradish peroxidase HRP-rabbit anti-mouse IgG (H+L) (Zymed Laboratories, USA). Proteins were then visualized with 1 min exposure to 1 ml of TrueBlue peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The purification of the fusion protein products expressed by recombinant E. coli BL21(DE) cells was performed using His·Bind kits (Novagen), following the provided protocol, and then the purified proteins were analyzed by SDS-PAGE.

The E. coli BL21 cells containing pET28a(+)-sllB were analyzed by electron microscopy (JEM-2010/INCA Oxford, Japan Electronics Co., Ltd./UK Oxford Company) at the instrumental analysis center of Shanghai Jiao Tong University. The 100 μl precipitate of E. coli BL21 cells containing pET28a(+)-sllB was harvested by centrifugation, then fixed by glutaraldehyde (2.5%) for 1 h. After two washings, the pellet was resuspended with 50 μl of PBS, followed by the addition of 5 μl of 2% phosphotungstic acid. One hour later, the pellet was washed twice more and resuspended in 50 μl of PBS. Finally, 20 μl of the final product was placed on copper mesh, allowed to dry, and observed under the lamps.

The sequenced results were edited to remove vector sequences and extra restriction sites, and then put into BLAST (Basic Local Alignment Search Tool) on GenBank to determine the identity of homologous sequence. The ORF was obtained using the ORF finder on the NCBI (National Center for Biotechnology Information) website. The amino acid sequence of the S-layer was obtained using Translate Tools in the ExPaSy web server, its physicochemical properties were analyzed by ProtParam Tools, and the signal peptide sequence examined by SignalP 3.0 software, hydrophilicity was analyzed by ProtScale tools, secondary structure by GOR4.0, and the functional site by InterProScan, ScanProsite, PPSearch and PROSITE software.

Using genomic DNA as a template, the gene fragment sllB was amplified by PCR with primers designed according to the published sequence (GenBank accession no. AJ849550), then 5 μl of the product was analyzed by agarose gel electrophoresis (Fig. 1). The PCR product was recovered and inserted into a pMD19-T simple vector to create the recombinant plasmid pMD19-T-sllB. This was then transformed into E. coli JM109 Competent Cells. Positive cells were selected by blue/white screening on LB plates and inserts were sequenced. According to Blastn in GenBank, our sequenced result had a 98.89% identity match with then B. sphaericus (strain JG-A12) partial gene for S-layer protein (GenBank no. AJ292965), a 97.57% identity match with B. sphaericus (NCTC JG-A12) slfB gene (GenBank no. AJ866975), a 97.46% identity match with B. sphaericus (strain JG-A12) sllB gene (GenBank no. AJ849550) for S-layer-like protein, a 91.61% identity match with B. sphaericus (strain JG-A12) slfB gene (GenBank no. AJ849549) for S-layer protein, a 90.30 % identity match with B. sphaericus (strain NCTC 9602) slfA gene (GenBank no. AJ866974), a 90.30% identity match with B. sphaericus (strain NCTC9602) sllA gene (GenBank no. AJ849548) for S-layer-like protein, and a 90.12% match with the B. sphaericus (strain NCTC9602) SllA pseudogene (GenBank no. AJ292964). We submitted our sequenced results to GenBank and received the number HM480488. One ORF was identified and the length of the sllB nucleic acid sequence was determined to be 3306 bp, after removal of the vector sequence and the added restriction sites.

The sllB cDNA fragment was excised from recombinant plasmid pMD19-T-sllB, sub-cloned into expression vector of pET28a (+), and confirmed by restriction enzyme analysis. E. coli BL21 was transformed with plasmid pET28a (+)-sllB and protein expression was induced with IPTG. A single band from SDS-PAGE (Fig. 2A) and western blotting (Fig. 2B) was observed on whole cell lysate, soluble protein fraction, and insoluble protein fraction, confirming the predicted molecular weight for the S-layer protein. E. coli BL21 carrying pET28a (+)-sllB were recovered from 2L of fermentation solution, collected, ultrasonically disrupted, and centrifuged. The precipitate was washed, dissolved, and filtered, and the supernatant purified by nickel-affinity chromatography, eluted with imidazole solution, and visualized in SDS-PAGE (Fig. 3). The elution fractions were collected, freeze-dried, and 1.8 mg of protein power obtained.

A few proteins were observed by electron microscopy on the surface of E. coli BL21 cells transformed with pET28a(+)-sllB and expressing the recombinant protein rS-layer (Fig. 4). The monolayer consisted of numerous randomly-orientated patches, clearly exhibiting the square lattice structure. The proteins were located on the surface of E. coli BL21 cells, and exogenous proteins were also present on E. coli BL21 cells.

In order to forecast the physicochemical property of recombinant S-layer proteins, the amino acid sequence of the rS-layer was derived from its nucleotide sequence. The deduced pre-protein was found to be composed of 1101 amino acid residues, with a molecular mass of 116.2613 kDa and a theoretical pI of 5.40. By ProtParam, the instability index (II) of the complete prepro-form protein was computed to be 12.25, which classified the protein as stable. The obtained grand average of hydropathicity (GRAVY) was −0.108, demonstrating that this protein was hydrophobic, confirming a previous prediction by ProScale software in the ExPaSy web server. The most likely cleavage site was between amino acid positions 31 and 32.

After this signal peptide sequence was removed, the mature protein was composed of 1070 residues, consisting of Ala (119aa, 11.1%), Arg (20aa, 1.9%), Asn (79aa, 7.4%), Asp (56aa, 5.2%), Gln (35aa, 3.3%), Glu (57aa, 5.3%), Gly (78aa,7.3%), His (5aa, 0.5%), Ile (53aa, 5.0%), Leu (47aa, 4.4%), Lys (77aa, 7.2%), Met (3aa, 0.3%), Phe (37aa, 3.5%), Pro (30aa, 2.8% ), Ser (62aa, 5.8%), Thr (131aa, 12.2%), Trp (3aa, 0.3%), Tyr (36aa, 3.4%) and Val (142aa, 13.3%), with a molecular mass of 113130.6 Da and a theoretical pI of 5.22. Four different software programs, InterProScan, ScanProsite, PPSearch and PROSITE, were used to predict the specific motifs of the recombinant protein. All of these analyses showed consistent results of three S-layer homology (SLH) domains in this mature protein, positioned at 1–61aa, 63–128aa and 137–197aa, respectively. The secondary structure of the mature S-layer was predicted using the GOR4 software and the results showed that 24.86% (266 amino acids) of the protein were alpha helices, 27.01% (289 amino acids) were extended strands, and 48.13% (515 amino acids) were random coils (Fig. 5).