Increasing the buffering capacity of minimal media leads to higher protein yield

Abstract

We describe a general and simple modification to the standard M9 minimal medium recipe that leads to an approximate twofold increase in the yield of heterologously expressed proteins in E. coli BL21(DE3) bacteria. We monitored the growth of bacteria transformed with plasmids for three different test proteins in five minimal media with different concentrations of buffering salts and/or initial media pH. After purification of the over-expressed proteins, we found a clear correlation between the protein yield and change in media pH over time, where the minimal media that were the most buffered and therefore most resistant to change in pH produced the most protein. And in all three test protein cases, the difference in yield was nearly twofold between the best and worst buffering media. Thus, we propose that increasing the buffering capacity of M9 minimal media will generally lead to a similar increase for most of the proteins currently produced by this standard protein expression protocol. Moreover, we have qualitatively found that this effect also extends to deuterated M9 minimal media growths, which could lead to significant cost savings in these preparations.

A missense mutation in the CSTF2 gene that impairs the function of the RNA recognition motif and causes defects in 3′ end processing is associated with intellectual disability in humans

“…Expression and purification of the proteins over metal affinity resin and His-tag removal was as done before ( 22 ). For the NMR experiments, transfected Rosetta (DE3) pLysS cells were grown in 2× minimal M9 media using 15 NH 4 Cl (1 g/l) and unlabeled or uniformly 13 C-labeled d -glucose (3 g/l) as sole nitrogen and carbon sources, respectively ( 37 ). Induction of the transfected cells with 0.5 mM isopropyl-β- d -thiogalactopyranoside (IPTG), harvest and purification of the labeled proteins was also carried out as previously described ( 22 ).…”

A DNA aptamer reveals an allosteric site for inhibition in metallo-β-lactamases

“…Glycerol (20% v/v final concentration) was added to the pure enzyme, which was stored at -20 °C. For NMR experiments, isotopically labeled enzyme was purified in the same way, however, the cells were grown in 2x M9 minimal medium (13.6 g/L Na 2 HPO 4 , 6.0 g/L KH 2 PO 4 , 1.0 g/L NaCl, 0.5 g/L Mg 2 SO 4 ∙7H 2 O, pH 7.1) [32] supplemented with 12 C- or 13 C- d -glucose (3 g/L) and ( 15 NH 4 ) 2 SO 4 (1 g/L) as the sole carbon and nitrogen sources, respectively, along with vitamins (1 μg/mL each biotin and thiamine) and kanamycin (50 μg/ml). Generally, ~40 and ~25 milligrams of purified enzyme were produced from per liter LB or minimal media, respectively.…”

NMR Lineshape Analysis of Intrinsically Disordered Protein Interactions

“…The protein to be observed should be prepared with uniform 15 N labelling, according to standard expression protocols [ 47 – 49 ]. Although resonance assignments are not strictly necessary for the analysis of titration data, they greatly assist the interpretation of the results, and therefore if assignments are not already available, it is recommended to prepare a second sample with 13 C/ 15 N labelling in order to carry out a backbone assignment according to standard methods [ 50 , 51 ].…”