Selectively Labeling the Heterologous Protein in Escherichia coli for NMR Studies: A Strategy to Speed Up NMR Spectroscopy

Screening methods to determine biophysical properties of proteins in structural genomics

“…In such cases we suggest that the digestion step should be done directly on the lysate. Since this prevents removal of the cellular proteins, the background protein level may be a problem but there are methods to avoid interfering signals from cellular proteins [6,7]. Batch cleavage instead of oncolumn cleavage might also be feasible even without any attempts to decrease the background protein level (top panel in Fig.…”

“…There have also been reports on different labeling schemes to enhance the labeling of the target, keeping the background proteins unlabeled or labeled at a lower level. Addition of rifampicin at the time for induction of expression in connection with a change from unlabeled to labeled medium is a way to increase the specificity of the labeling of overproduced protein and has been shown to yield NMR data of good quality [6]. Specific amino acid labeling has also been suggested as a way to reduce the need for sample purification [7].…”

Important Contributions of a New Quantitative Preparative Native Continuous Polyacrylamide Gel Electrophoresis (QPNC-PAGE) Procedure for Elucidating Metal Cofactor Metabolisms in Protein-Misfolding Diseases - A Theory

“…It demands high protein concentrations and high purity [36]. However, it is also possible to obtain NMR spectra at sub-micromolar protein concentrations or at even lower concentrations of analytes [37].…”

Site-specific Reflex Response of Ubiquitin to Loop Insertions

“…The supernatant was brought to 10% 2 H 2 O and HSQC spectra were taken with no further purification. 34 Protein expression and purification BL21(DE3)* E. coli cells were transformed, grown in 1 l of 15 N-labeled media, and protein expression was induced as described above. The cells were pelleted, resuspended in 15 ml of 50 mM Tris (pH 7.65), 10 mM EDTA, 1 mM DTT, and lysed by sonication.…”