Large-Scale Production and Purification of MelBSt Protein

Cell growth for the large-scale production of WT MelB_St was carried out using the pK95 ΔAH/MelB_St/CHis₁₀ from E. coli DW2 cells (melA⁺melB, lacZY) as described (10 (link), 39 (link)). Briefly, MelB_St purification from membranes by cobalt-affinity chromatography (Talon Superflow Metal Affinity Resin, Takara) after extraction by 1.5% UDM or 2% DDM. MelB_St protein was eluted with 250 mM imidazole in a buffer containing 50 mM NaPi, pH 7.5, 200 mM NaCl, 0.035% UDM or 0.01% DDM, and 10% glycerol, and dialyzed to change the buffer conditions accordingly.

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Katsube S., Willibal K., Vemulapally S., Hariharan P., Tikhonova E., Pardon E., Kaback H.R., Steyaert J, & Guan L. (2023). In vivo and in vitro characterizations of melibiose permease (MelB) conformation-dependent nanobodies reveal sugar-binding mechanisms. The Journal of Biological Chemistry, 299(8), 104967.

Publication 2023

Talon Superflow Metal Affinity Resin

Affinity chromatography Buffer Cobalt E coli Glycerol Imidazole Mela Membranes Metal Nacl Protein Resin Talon

Corresponding Organization : Texas Tech University

Other organizations : Vrije Universiteit Brussel, VIB-VUB Center for Structural Biology, University of California, Los Angeles

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Variable analysis

independent variables

Extraction method (1.5% UDM or 2% DDM)

dependent variables

MelB_St protein purification yield

control variables

Protein source (pK95 ΔAH/MelB_St/CHis10 from E. coli DW2 cells (melA+ melB, lacZY))
Cobalt-affinity chromatography (Talon Superflow Metal Affinity Resin, Takara) for MelB_St purification
Elution buffer (250 mM imidazole in a buffer containing 50 mM NaPi, pH 7.5, 200 mM NaCl, 0.035% UDM or 0.01% DDM, and 10% glycerol)
Dialysis to change buffer conditions

controls

No positive or negative controls were explicitly mentioned.

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