Chapter Three - Escherichia coli Auxotroph Host Strains for Amino Acid-Selective Isotope Labeling of Recombinant Proteins
Introduction
Amino acid-selective isotope labeling of proteins is a powerful approach in structural and functional studies of proteins. Enrichment of proteins at selected residue types with 15N and/or 13C isotopes greatly simplifies the chemical shift assignments in nuclear magnetic resonance (NMR) spectroscopic studies (Verardi, Traaseth, Masterson, Vostrikov, & Veglia, 2012). We have also employed similar approaches to characterize the interactions between enzymes and their cofactors such as a semiquinone and an iron–sulfur cluster by pulsed electron paramagnetic resonance (EPR) spectroscopy (Iwasaki et al., 2012, Iwasaki et al., 2009, Lin et al., 2012, Lin et al., 2008). In addition, vibrational spectroscopies such as resonance Raman and Fourier transform infrared also greatly benefit from specific isotope labeling in determining protein–ligand and protein–protein interactions (Haris, 2010, Rotsaert et al., 2003). All these techniques provide deeper mechanistic insights into the functions of proteins that are often not readily accessible from their crystal structures.
Escherichia coli has been the most commonly used host for heterologous production of foreign proteins due to its straightforward and inexpensive cultivation, well-known genetics, and highly developed biotechnological tools (Zerbs, Frank, & Collart, 2009). If a protein of interest can be successfully expressed in E. coli and purified in its functional forms, using E. coli for selective amino acid isotope labeling is likely the most reasonable and cost-effective option. Similarly, selective unlabeling of proteins with individual amino acids or metabolic precursors may also be useful in NMR peak assignments (Bellstedt et al., 2013, Rasia et al., 2012). However, E. coli possesses biosynthetic and biodegradation networks for all 20 amino acids (Waugh, 1996), and these pathways intersect in a complex manner such that one amino acid can be readily converted into other types. The result is often the dilution and scrambling of the input isotope label to undesired amino acid types.
The extent of metabolic scrambling in E. coli depends on not only the amino acid types and the location of the isotopic label (Waugh, 1996) but also the bacterial growth conditions. For example, simply adding individual 15N- and/or 13C-labeled histidine, lysine, or methionine in the growth media will effectively enrich proteins labeled at the intended amino acids when cultured in the growth medium supplied with other unlabeled amino acid types (Iwasaki et al., 2009, O'Grady et al., 2012). However, attempting this with most other amino acids under the same conditions results in significant dilution and scrambling of the isotope labels (O'Grady et al., 2012). 15Nα labels, commonly used in NMR experiments for chemical shift assignments, are specifically readily transferred from one amino acid type to another by any of the four aminotransferases present in E. coli (encoded by aspC, avtA, ilvE, and tyrB) (Waugh, 1996). These problems can be minimized by genetic engineering of the host strains to optimize for the desired label distribution.
Section snippets
E. coli Auxotrophs for Amino Acid-Selective Isotope Labeling
The approach is to produce proteins in E. coli auxotrophic strains in which enzymes that result in dilution and scrambling of the label (Waugh, 1996) have been eliminated, avoiding mutations or groups of mutations that would result in impeding cell growth. A set of isogenic auxotrophic host strains has been generated using E. coli parent strains, BL21(DE3) or C43(DE3), which are designed for the widely used T7 RNA polymerase overexpression system to enhance their utility. These strains (Table 1
Methods
In general, optimal conditions for the overexpression of proteins in E. coli can vary considerably (Zerbs et al., 2009). For high-level production of simple and complex proteins with bound cofactors required for biophysical studies, the following factors may be considered:
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Coupling efficiency of the apoprotein translation and folding speeds with those of cofactor insertion and/or processing may be tuned by the growth rate of the E. coli expression host strains by temperature control, e.g., at
Conclusions
Amino acid-selective isotope labeling is a powerful tool required to make full use of magnetic resonance and vibrational spectroscopies as applied to protein structure and dynamics. For this purpose, we have generated a set of BL21(DE3) or C43(DE3)-derived E. coli amino acid auxotrophic expression host strains (Table 1) (Iwasaki et al., 2012, Lin et al., 2011), which can help minimize possible metabolic scrambling of the input isotopic labels of many different amino acid types. Examples of the
Acknowledgments
This auxotroph strain bank project was supported in part by the JSPS-NSF International Collaborations in Chemistry (ICC) Grant from JSPS (T.I.), the JSPS Grant-in-aid 24659202 (T.I.), and the Nagase Science and Technology Foundation Research Grant (T.I.). Additional funding was provided by the DE-FG02-87ER13716 (R.B.G.) Grant from Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, U.S. Department of Energy, and from the National
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Current address: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.