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 over-expression 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-a-vis NMR spectroscopy.
Highlights
The two most popular methods today for structural studies of biomolecular systems are X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy
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
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
Summary
The two most popular methods today for structural studies of biomolecular systems are X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy. Bacterial expression parameters to be optimized in this method to attain a very high cell density in laboratory shaking cultures are: 1) double selection of colonies highly expressing the target protein; 2) optimization of temperature and time period for starter culture to avoid plasmid instability or loss; 3) optimizing induction temperature and time course; and 4) glucose optimization. To overcome decreased rate of incorporation of appropriate isotopes, a longer period of clearance of unlabeled nutrients is allotted before induction It favors metabolic rates of E. coli in deuterated media [11] and high yield expression. A lower temperature for prolonged duration of time is preferred to obtain soluble target protein at high yield without any additional requirement of amino acids and vitamin supplements [9]
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