For expression of SeMet-labeled RovC, transformed cells were first grown in 2 x 500 ml of DYT medium (supplemented with 30 μg/ml kanamycin and 34 μg/ml chloramphenicol) at 37°C, 130 rpm overnight. 500 ml of fresh DYT medium (supplemented with 30 μg/ml kanamycin and 34 μg/ml chloramphenicol) was added to each of the overnight cultures and cells were grown for another hour at 130 rpm, 37°C. Cells were harvested by centrifugation at 6000 rpm, 4°C for 5 mins and washed twice with minimal medium (per liter: 1 g NH4Cl, 3 g KH2PO4, 4 g Na2HPO4, 22 g glucose, 0.61 g MgSO4, 11.2 mg thiamine-HCl, 10.4 mg Fe2(SO4)3•7 H2O) by centrifugation at 6000 rpm, 5 min at 4°C. The pellet was resuspended in minimal medium and used to inoculate 4 L of Se-Met Medium (minimal medium + 50 mg/l of selenomethionine (Acros Organics)) to an OD600 of 1.0. Induction of expression, cell harvest and protein purification were performed as described above.
Selenomethionine
Manufactured by Thermo Fisher Scientific
Selenomethionine is a form of the essential amino acid methionine, in which the sulfur atom is replaced by a selenium atom. It is a common nutritional supplement and is used in various biochemical and biomedical applications.
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5 protocols using selenomethionine
1
Expression and Purification of SeMet-RovC
2
Recombinant suPAR Production in Drosophila
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Recombinant suPAR (residue 1–277) were secreted from Drosophila S2 cells as previously described59 . The secreted protein was captured from the conditioned medium using an ATF affinity column followed by reversed-phase HPLC using a C4 column on the HPLC system. The fractions containing the pure protein were lyophilized.
To produce the selenomethionine (SeMet)-substituted suPAR, stable suPAR S2 cells were grown to 2L and reached a concentration of 10–12 × 106 cells/mL in EX-CELL 420 serum-free medium. The cells were then washed in a methionine-free medium (Orbigen) and starved in this medium for 4–6 h at 28 °C before 60 μg/mL selenomethionine (Acros Organics) was added to the culture. The protein expression was induced with the addition of 500 μM of CuSO4. The culture medium was harvested 4–5 days after induction. The purification was similar to that of the native protein. Amino acid analysis showed that 90% of native methionine in the protein had been substituted by SeMet.
To produce the selenomethionine (SeMet)-substituted suPAR, stable suPAR S2 cells were grown to 2L and reached a concentration of 10–12 × 106 cells/mL in EX-CELL 420 serum-free medium. The cells were then washed in a methionine-free medium (Orbigen) and starved in this medium for 4–6 h at 28 °C before 60 μg/mL selenomethionine (Acros Organics) was added to the culture. The protein expression was induced with the addition of 500 μM of CuSO4. The culture medium was harvested 4–5 days after induction. The purification was similar to that of the native protein. Amino acid analysis showed that 90% of native methionine in the protein had been substituted by SeMet.
3
Selenomethionine-labeled PupR CCSSD Expression
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Selenomethionine (Acros Organics)-derivatized PupR CCSSD was expressed using a modified protocol involving methionine synthesis suppression (29 (link), 30 (link)). E. coli C41(DE3) cells transformed with pMBP-Parallel1-PupR CCSSD were grown at 37 °C to saturation in 3 ml of LB medium with 100 μg/ml of ampicillin, then transferred to pre-warmed M9 minimal medium containing 2 mm MgSO4, 0.1 mm CaCl2, 0.4% (w/v) glucose, and 100 μg/ml of ampicillin and incubated at 37 °C. Once the OD600 nm reached 1.0, the medium was supplemented with Se-Met, Lys, Thr, Phe, Leu, Ile, and Val; and the temperature lowered to 20 °C. Protein expression was induced with 0.5 mm isopropyl 1-thio-β-d -galactopyranoside for 18 h. Purification of Se-Met PupR CCSSD was performed as described for native protein. The molecular mass of the final protein samples and Se-Met incorporation were confirmed by electrospray ionization MS.
4
Selenomethionine-Labeled VSIG3 Protein
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In order to obtain phase information and analyze the final crystal structure, E. coil cells were cultured in M9-deficient medium (Molecular Dimensions), and selenomethionine (Acros Organics) was added in the process of VSIG3 expression. The methionine in the final expressed VSIG3 protein was replaced by seleno methionine.
5
Recombinant suPAR Protein Production
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Recombinant suPAR (residue 1-277) were secreted from Drosophila S2 cells as previously described 53 .
The secreted protein was captured form the conditioned medium using ATF a nity column followed by reversed-phase HPLC using a C4 column on HPLC system. The fractions containing the pure protein was lyophilized.
To produce the selenomethionine (SeMet)-substituted suPAR protein, stable suPAR S2 cells were grown to 2 L and reached a concentration of 10-12×10 6 cells/ml in EX-CELL 420 serum-free medium. The cells were then washed in methionine-free medium (Orbigen) and starved in this medium for 4-6 hr at 28°C before 60 µg/ml selenomethionine (Acros Organics) was added to the culture. The protein expression was induced with the addition of 500 µM of CuSO4. The culture medium was harvested 4-5 days after induction. The puri cation was similar to that of the native protein. Amino acid analysis showed that 90% of native methionine in the protein had been substituted by SeMet.
The secreted protein was captured form the conditioned medium using ATF a nity column followed by reversed-phase HPLC using a C4 column on HPLC system. The fractions containing the pure protein was lyophilized.
To produce the selenomethionine (SeMet)-substituted suPAR protein, stable suPAR S2 cells were grown to 2 L and reached a concentration of 10-12×10 6 cells/ml in EX-CELL 420 serum-free medium. The cells were then washed in methionine-free medium (Orbigen) and starved in this medium for 4-6 hr at 28°C before 60 µg/ml selenomethionine (Acros Organics) was added to the culture. The protein expression was induced with the addition of 500 µM of CuSO4. The culture medium was harvested 4-5 days after induction. The puri cation was similar to that of the native protein. Amino acid analysis showed that 90% of native methionine in the protein had been substituted by SeMet.
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