Supplementary Material
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Supporting information
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Supplementary Methods
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Supplementary Fig. 1 – Schematic representation of the amino acid sequence of BA (NCBI
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reference sequence; BAA20465).
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Supplementary Fig. 2 – (a) Overall scheme of the primary structures of GRP_BA and
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recombinant GRP_BA protein (rBA); (b) the optimized sequence of GRP_BA without N-
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terminal signal sequence for over-expression.
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Supplementary Fig. 3 – Wide scan X-ray photoelectron spectrum (XPS) of CaCO3 precipitates
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obtained at 4 °C after a 20 h incubation using the ammonium carbonate vaporization method (V-
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L) with 1 mg rBA ml−1.
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Strains and vector construction
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E. coli DH5α (Life Technologies, Carlsbad, CA, USA) cell line was used as a host for
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recombinant vector preparation, and E. coli BL21 (DE3) (Merck) was used for the expression of
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rBA. The E. coli cells were grown in Luria-Bertani (LB) medium with 50 µg ampicillin ml-1
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(Sigma-Aldrich). The amino acid sequence of rBA (NCBI reference sequence BAA20465) was
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obtained from an NCBI BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The rBA gene
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sequence was redesigned based on E. coli codon preference and to avoid gene sequence repeats
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and then chemically synthesized (GenScript USA Inc., Piscataway, NJ, USA). Using NdeI and
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XhoI restriction sites, the sequence was introduced into the pET23b+ vector (Novagen,
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Darmstadt, Germany), which contains a strong T7 promoter and six-histidine (His6) tag. Finally,
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the vector construct was confirmed by direct sequencing.
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Over-expression and purification of rBA
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The vector construct was introduced into E. coli BL21 (DE3) cells by electroporation. A single
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colony from a freshly streaked plate of these cells was grown in 400 ml of LB medium with 50
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µg ampicillin
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thiogalactopyranoside (IPTG; 0.2 mM final concentration; Sigma-Aldrich) was added to induce
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the protein over-expression, and the cells were further incubated at 37 ºC and 200 rpm for 4 h.
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The cells were harvested by centrifugation at 8,911 g for 10 min at 4 ºC, and the cell pellets were
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stored at -80 ºC.
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For Ni-NTA affinity purification, The cells were resuspended in 40 ml of lysis buffer (50 mM
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NaH2PO4, 300 mM NaCl and 10 mM imidazole; pH 8.0). The cells were lysed using an
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ultrasonic cell disrupter (ULH-700S; Ulsso High-Tech Co., Cheongwon, Korea) at 30% power
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with a 3 sec on pulse and a 10 sec cooling period between each burst. The lysate was centrifuged
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at 8,911 g for 10 min at 4 ºC. The insoluble pellet was collected, washed twice and resuspended
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in 5 ml of denaturing lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole and 8 M
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urea; pH 8.0) per gram of wet weight. The supernatant was collected and applied to Ni-
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nitrilotriacetic acid (Ni-NTA) resin (Qiagen, Germantown, MD, USA) for affinity purification.
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After incubating the lysate-Ni-NTA mixture at 4 ºC for 1 h, the mixture was loaded onto a
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column. The resin was washed three times with five column volumes of denaturing wash buffer
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(50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole and 8 M urea; pH 8.0). The rBA protein
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was eluted using elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM and imidazole; pH
ml-1 at 37 ºC and 200 rpm until the OD600 was 0.8-1.0. Isopropyl-β-D-
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8.0). The eluted rBA protein was loaded into a dialysis membrane bag with a 12-14 kDa
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molecular weight cut off (Spectrum Laboratories Inc., Los Angeles, CA, USA). The sample was
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placed into 3L of external chamber of distilled water with gentle stirring and dialyzed for about
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12 h (overnight), and the dialysis buffer was subsequently changed three times. Finally, the
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purified rBA protein was freeze-dried and stored at -80 ºC.
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In addition, inclusion body purification was conducted, including lipopolysaccharides removal
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and non-ionic surface treatment, based on a previously described method with the following
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modifications (Choi et al. 2014). Harvested cells were sequentially resuspended and washed with
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washing solution I (100 mM Tris-HCl, pH 8.0), washing solution II (5 mM CaCl2, 100 mM Tris-
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HCl, pH 8.0) and washing solution III (10 mM EDTA, 100 mM Tris-HCl, pH 8.0). The
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procedure was repeated three times, and cell pellets were resuspended in 15 ml washing solution
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I per gram wet weight. Cells were lysed with a tabletop laboratory homogenizer system
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(PandaPLUS, GEA Group, Germany), and inclusion bodies were collected by centrifugation at
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8,911 g for 10 min at 4 ºC. The inclusion bodies were washed twice with TTE buffer I (1%
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Triton X-100, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0) and TTE buffer II (1% Triton X-100, 50
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mM Tris-HCl, pH 8.0). Then, the inclusion bodies were solubilized using 8 M urea and below 20
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ml of the solubilized rBA protein solution was loaded into a dialysis membrane bag with a 12-14
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kDa molecular weight cut off. The sample was placed into 3L of external chamber of distilled
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water with gentle stirring and dialyzed for about 12 h (overnight), and the dialysis buffer was
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subsequently changed three times. Urea removal was monitored by measuring the UV
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absorbance of the external solution at 200 nm. Finally, the purified rBA protein was freeze-dried
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and stored at -80 ºC. The expression level and the purity were monitored by sodium dodecyl
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sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein concentration was
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assessed using the bicinchoninic acid (BCA) assay method (Sigma-Aldrich).
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Supplementary Figures
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Supplementary Fig. 1. Schematic representation of the amino acid sequence of BA (NCBI
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reference sequence; BAA20465). Black underline indicates a signal peptide. The yellow box is
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the shematrin-2-like domain and the green box is a potential calcium-binding domain. Red letters
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indicate acidic amino acid residues and blue letters indicate basic amino acid residues.
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Supplementary Fig. 2. (a) Overall scheme of the primary structures of GRP_BA and
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recombinant GRP_BA protein (rBA); (b) the optimized sequence of GRP_BA without N-
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terminal signal sequence for over-expression.
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Supplementary Fig. 3. Wide scan X-ray photoelectron spectrum (XPS) of CaCO3 precipitates
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obtained at 4 °C after a 20 h incubation using the ammonium carbonate vaporization method (V-
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L) with 1 mg rBA ml-1.
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