High yield expression of proteins in <i>E. coli</i> for NMR studies

Abstract

In recent years, high yield expression of proteins in E. coli has witnessed rapid progress with developments of new methodologies and technologies. An important advancement has been the development of novel recombinant cloning approaches and protocols to express heterologous proteins for Nuclear Magnetic Resonance (NMR) studies and for isotopic enrichment. Isotope labeling in NMR is necessary for rapid acquisition of high dimensional spectra for structural studies. In addition, higher yield of proteins using various solubility and affinity tags has made protein overexpression cost-effective. Taken together, these methods have opened new avenues for structural studies of proteins and their interactions. This article deals with the different techniques that are employed for over-expression of proteins in E. coli and different methods used for isotope labeling of proteins vis-à-vis NMR spectroscopy.

A unique homodimeric NAD + ‐linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri

“…In addition, its affinity to isocitrate ( S 0.5 < 10 μM) is much higher than those of eukaryotic heteromeric NAD‐IDHs, and NADPH is not a noncompetitive inhibitor of OtIDH with respect to NAD + , unlike C. reinhardtii NAD‐IDH (2). Although 6His‐tag has the potential to interfere with structure or function of proteins, such as their mosaicity and diffraction, the small size and charge of the polyhistidine tag ensure that protein activity is rarely affected, except in a few instances (41). Therefore, OtIDH may represent a new type of NAD + ‐dependent IDH that is distinctly different from all known NAD‐IDHs in the type I subfamily.…”

Late metabolic precursors for selective aromatic residue labeling

“…After cellular uptake, these compounds are converted into the target residues within their metabolic pathways in-vivo. Such overexpression systems have been described for prokaryotic ( E. coli ) (Hoogstraten and Johnson 2008 ; Mondal et al 2013 ), as well as eukaryotic cells (yeast, insect cell-lines) (Morgan et al 2000 ; Takahashi and Shimada 2010 ). Especially when early metabolic intermediates are used as labeled nutrients, the danger of cross-labeling to unwanted positions is very high, thus resulting in unselective isotope patterns.…”

The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli

“…Mainly mineral media are used. Especially when producing proteins in minimal medium for subsequent structural characterization by NMR, poor growth, low levels of expression, and insufficient labeled protein are very often problematic [ 99 ]. The application of an intelligent growth system can solve this problem by addition of a 15 N labelled ammonium salt [ 100 , 101 ].…”