Strep-tagged GlnR overexpression in S. coelicolor M145 and purification

For Strep-GlnR purification, S. coelicolor M145 carrying pGM-Strep-glnR overexpression strain (Table ​(Table1)1) was grown for 4 days in complex S-medium at 30°C. Subsequently, cells were harvested and washed twice with nitrogen-free Evans medium. Washed cell biomass was transferred into Evans medium supplemented with 5 or 100 mM NH₄Cl and 5 or 100 mM NaNO₃ as a sole nitrogen source, respectively. The expression of Strep-GlnR was induced with 12.5 μg/ml thiostrepton for 36 h. After cultivation cells were harvested and washed with a solution of 100 mM Tris and 150 mM NaCl (pH 8.0). In order to prevent phosphatase and protease activity, 5 mM sodium fluoride and 5 mM orthovanadate and the EDTA-free cOmplete protease inhibitor cocktail (Roche) were added to the buffer. Cell lysis was performed by Emulsifex (Avestin, Ottawa, Canada) with three consecutive passages. Cell debris and insoluble proteins were separated from the soluble fraction by centrifugation (60 min, 14800 g, 4°C). Soluble proteins were loaded onto a pre-equilibrated Gravity flow Strep-Tactin^®; Sepharose^®; column for one-step purification of recombinant Strep-tag^®; proteins (1-ml bed volume; IBA, Germany). Strep-GlnR was competitively eluted using elution buffer supplemented with 2.5 mM desthiobiotin. Concentrated fractions containing the pure Strep-GlnR were stored at 4°C.