The genes for the full-length LysGH15 and its three individual domains (i.e., CHAP, amidase-2, and SH3b) were amplified using corresponding primers that were designed based on the full-length lysGH15 gene (GenBank: AY176327) and were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The coding regions for the CHAP (residues 1–165), amidase-2 (residues 165–403), and SH3b (residues 368–495) domains were cloned into the pMCSG7 vector as previously reported [41] (link). The full-length lysGH15 gene was subcloned into the pET-26b vector. Mutations were designed based on these constructs and were generated using the QuikChange Site-Directed Mutagenesis Kit following the manufacturer’s instructions (Stratagene). All of the recombinant plasmids were sequenced to verify the sequence.
The plasmids harboring the target gene, which encoded 6× His-tagged proteins, were transformed into E. coli BL21(DE3) (Tiangen Biotechnology). The cells were grown in Luria-Bertani (LB) medium at 37°C until the OD600 reached 0.8. The culture was then induced with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) for 20 h at 16°C. Cells were harvested by centrifugation at 4,670×g for 30 min and were resuspended in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 50 mM Na2HPO4, and 10 mM KH2PO4, pH 7.4). After lysis by sonication, the cell debris was removed by centrifugation at 38,900×g for 30 min. The supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) resin gravity column (Qiagen) that had been previously equilibrated with PBS. The column was washed using 100 ml of lysis buffer containing 20 mM imidazole, followed by a 50 mM imidazole wash. Finally, the protein was eluted with PBS containing 500 mM imidazole. After buffer exchange, the 6× His-tag was removed using tobacco etch virus (TEV) proteolysis (except for full-length LysGH15). Uncut protein was removed using a second Ni-affinity chromatography step. The proteins without a His-tag were concentrated and applied to a Superdex G200 size-exclusion chromatography column (Amersham) that was preequilibrated with 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl (500 mM NaCl for full-length LysGH15). For the SH3b domain, 40 mM Na3PO4 and 50 mM NaCl, pH 6.5, were used. Fractions containing the purified target protein were pooled and stored at −80°C until further analysis.
The E. coli BL21(DE3) strain that contained the pMCSG7-CHAP vector was grown in M9 medium containing glucose (0.2% M/V), MgSO4 (1 mM), and ampicillin (100 µg/ml) at 37°C until the OD600 reached 0.8. Subsequently, selenomethionine was added to the culture (50 µg/ml). The subsequent purification steps were similar to those used for the native protein.
The plasmid pMCSG7-SH3b was transformed into E. coli BL21(DE3). The cells were grown in M9 medium containing glucose (0.2% M/V), MgSO4 (1 mM), and ampicillin (100 µg/ml). 15N ammonium chloride and/or 13C glucose was used as the sole nitrogen and carbon sources, respectively, for isotope labeling. Labeled SH3b was purified using an identical procedure as that used for the native protein.
The plasmids harboring the target gene, which encoded 6× His-tagged proteins, were transformed into E. coli BL21(DE3) (Tiangen Biotechnology). The cells were grown in Luria-Bertani (LB) medium at 37°C until the OD600 reached 0.8. The culture was then induced with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) for 20 h at 16°C. Cells were harvested by centrifugation at 4,670×g for 30 min and were resuspended in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 50 mM Na2HPO4, and 10 mM KH2PO4, pH 7.4). After lysis by sonication, the cell debris was removed by centrifugation at 38,900×g for 30 min. The supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) resin gravity column (Qiagen) that had been previously equilibrated with PBS. The column was washed using 100 ml of lysis buffer containing 20 mM imidazole, followed by a 50 mM imidazole wash. Finally, the protein was eluted with PBS containing 500 mM imidazole. After buffer exchange, the 6× His-tag was removed using tobacco etch virus (TEV) proteolysis (except for full-length LysGH15). Uncut protein was removed using a second Ni-affinity chromatography step. The proteins without a His-tag were concentrated and applied to a Superdex G200 size-exclusion chromatography column (Amersham) that was preequilibrated with 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl (500 mM NaCl for full-length LysGH15). For the SH3b domain, 40 mM Na3PO4 and 50 mM NaCl, pH 6.5, were used. Fractions containing the purified target protein were pooled and stored at −80°C until further analysis.
The E. coli BL21(DE3) strain that contained the pMCSG7-CHAP vector was grown in M9 medium containing glucose (0.2% M/V), MgSO4 (1 mM), and ampicillin (100 µg/ml) at 37°C until the OD600 reached 0.8. Subsequently, selenomethionine was added to the culture (50 µg/ml). The subsequent purification steps were similar to those used for the native protein.
The plasmid pMCSG7-SH3b was transformed into E. coli BL21(DE3). The cells were grown in M9 medium containing glucose (0.2% M/V), MgSO4 (1 mM), and ampicillin (100 µg/ml). 15N ammonium chloride and/or 13C glucose was used as the sole nitrogen and carbon sources, respectively, for isotope labeling. Labeled SH3b was purified using an identical procedure as that used for the native protein.
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