For NMR structure determination of the eight selected designs, uniformly isotope-labeled [U-15N, U-13C] proteins were expressed using the method described above, except that [13C]glucose was used as a sole carbon source. The [U-15N, U-13C]-enriched proteins were purified through a Ni-NTA affinity column followed by gel filtration chromatography on an ÄKTA Pure 25 FPLC (GE Healthcare) using a Superdex 75 Increase 10/300 GL column (GE Healthcare). The purified proteins were dissolved in 95% 1H2O/5% 2H2O PBS buffer at various pH (50 mM NaCl, 1.1 mM Na2HPO4 and 7.4 mM KH2PO4 at pH 6.0 for NF2-02, NF3-03 and NF6-02; 50 mM NaCl, 4.3 mM Na2HPO4 and 5.7 mM KH2PO4 at pH 6.8 for NF5-03, NF7-04 and NF8-01; 50 mM NaCl, 5.6 mM Na2HPO4 and 1.1 mM KH2PO4 at pH 7.4 for NF1-14; and 137 mM NaCl, 1.1 mM Na2HPO4 and 7.4 mM KH2PO4 at pH 7.4 for NF4-04). Shigemi micro-NMR tubes were used for all NMR measurements except RDC (protein concentration ~900 μM for all designed proteins except NF4-04 (~400 μM) and NF6-02 (~700 μM)), and normal NMR tubes were used for RDC experiments (protein concentration ~200 μM).
Kta pure 25 fplc
Manufactured by GE Healthcare
The ÄKTA Pure 25 FPLC is a fast protein liquid chromatography (FPLC) system designed for protein purification and analysis. It is capable of performing a range of chromatographic techniques, including ion exchange, size exclusion, and affinity chromatography. The system features a flow rate of up to 25 mL/min and can handle sample volumes up to 50 mL. It is compatible with a variety of column sizes and can be operated using the intuitive UNICORN control software.
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2 protocols using kta pure 25 fplc
1
NMR Structure Determination of Designed Proteins
2
Optimized Expression and Purification of VAL88 Proteins
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Two-dimensional 1H–15N HSQC spectrum was measured to verify whether the core of VAL88 proteins is well packed, i.e., not in a molten globule state. The VAL88 was expressed in E. coli BL21 Star (DE3) cell as uniformly (U-)15N-labeled proteins using MJ9 minimal media (48 (link)), which contain 15N ammonium sulfate as a sole nitrogen source and 12C glucose as a sole carbon source, respectively. The expressed proteins with a 6×His tag at the C terminus were purified through a nickel affinity column and then purified by gel filtration chromatography on an ÄKTA Pure 25 FPLC (GE Healthcare) using a Superdex 75 increase 10/300 GL column (GE Healthcare). The expression, solubility, and purity of VAL88 proteins were assessed by SDS/PAGE and mass spectrometry (Thermo Scientific Orbitrap Elite). The HSQC spectrum was collected for 500 μM protein sample in 90% H2O/10% D2O buffer containing 100 mM NaCl, 1.2 mM Na2HPO4, and 7.4 mM KH2PO4 at pH 6.0, at 25 °C on a JEOL JNM-ECA 600-MHz spectrometer.
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