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A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.

Mehlhorn J, Steinocher H, Beck S, Kennis JT, Hegemann P, Mathes T - PLoS ONE (2013)

Bottom Line: For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties.Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation.Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie/Experimentelle Biophysik, Humboldt-Universität zu Berlin, Berlin, Germany.

ABSTRACT
Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

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Amino acid and cofactor specific isotope labeling using custom-made auxotrophic expression strains (A) in a high cell density fermentation setup (B).CmpX13 is rendered auxotrophic for selected amino acid and/or cofactor synthesis pathways. The resulting expression strains are cultivated under controlled conditions to achieve the highest cell density under complete consumption of unlabeled substrates. Subsequently labeled substrates are fed and protein production is induced.
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pone-0079006-g001: Amino acid and cofactor specific isotope labeling using custom-made auxotrophic expression strains (A) in a high cell density fermentation setup (B).CmpX13 is rendered auxotrophic for selected amino acid and/or cofactor synthesis pathways. The resulting expression strains are cultivated under controlled conditions to achieve the highest cell density under complete consumption of unlabeled substrates. Subsequently labeled substrates are fed and protein production is induced.

Mentions: Here, we describe a straight-forward approach for selective labeling of amino acid side chains and flavin cofactors of a heterologously expressed protein. This strategy employs the disruption of amino acid and flavin biosynthesis pathways in an E. coli expression strain using standard homologous recombination techniques (Figure 1A) [36]. Similar approaches have been reported earlier, in which either well known auxotrophic E. coli mutants were transformed into suitable expression strains [27] or well known expression strains were rendered auxotrophic [35]. Here we use the previously engineered expression strain CmpX13 [37] as a parent strain. It contains a constitutively expressed riboflavin uptake system, which makes it especially suitable for strong overexpression of flavoproteins. Furthermore we created enhanced riboflavin auxotrophic strains in analogy to the previously described strain CmpX131 [37], but with additional expression of flavokinases to lower the riboflavin supplementation requirement. These strains facilitate efficient in vivo flavin reconstitution, which is especially useful for proteins that cannot be classically reconstituted in vitro by unfolding and refolding in the presence of the cofactor analog [38,39]. Additionally, we describe how these strains can be used for highly efficient selective isotope labeling in a high cell density fermentation (HCDF) procedure (Figure 1B) [40]. Finally, we illustrate the potential of this approach by presenting FTIR light-minus-dark difference spectra of the Synechocystis BLUF photoreceptor Slr1694 (SyPixD) with selective flavin and protein labeling patterns.


A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.

Mehlhorn J, Steinocher H, Beck S, Kennis JT, Hegemann P, Mathes T - PLoS ONE (2013)

Amino acid and cofactor specific isotope labeling using custom-made auxotrophic expression strains (A) in a high cell density fermentation setup (B).CmpX13 is rendered auxotrophic for selected amino acid and/or cofactor synthesis pathways. The resulting expression strains are cultivated under controlled conditions to achieve the highest cell density under complete consumption of unlabeled substrates. Subsequently labeled substrates are fed and protein production is induced.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815312&req=5

pone-0079006-g001: Amino acid and cofactor specific isotope labeling using custom-made auxotrophic expression strains (A) in a high cell density fermentation setup (B).CmpX13 is rendered auxotrophic for selected amino acid and/or cofactor synthesis pathways. The resulting expression strains are cultivated under controlled conditions to achieve the highest cell density under complete consumption of unlabeled substrates. Subsequently labeled substrates are fed and protein production is induced.
Mentions: Here, we describe a straight-forward approach for selective labeling of amino acid side chains and flavin cofactors of a heterologously expressed protein. This strategy employs the disruption of amino acid and flavin biosynthesis pathways in an E. coli expression strain using standard homologous recombination techniques (Figure 1A) [36]. Similar approaches have been reported earlier, in which either well known auxotrophic E. coli mutants were transformed into suitable expression strains [27] or well known expression strains were rendered auxotrophic [35]. Here we use the previously engineered expression strain CmpX13 [37] as a parent strain. It contains a constitutively expressed riboflavin uptake system, which makes it especially suitable for strong overexpression of flavoproteins. Furthermore we created enhanced riboflavin auxotrophic strains in analogy to the previously described strain CmpX131 [37], but with additional expression of flavokinases to lower the riboflavin supplementation requirement. These strains facilitate efficient in vivo flavin reconstitution, which is especially useful for proteins that cannot be classically reconstituted in vitro by unfolding and refolding in the presence of the cofactor analog [38,39]. Additionally, we describe how these strains can be used for highly efficient selective isotope labeling in a high cell density fermentation (HCDF) procedure (Figure 1B) [40]. Finally, we illustrate the potential of this approach by presenting FTIR light-minus-dark difference spectra of the Synechocystis BLUF photoreceptor Slr1694 (SyPixD) with selective flavin and protein labeling patterns.

Bottom Line: For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties.Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation.Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie/Experimentelle Biophysik, Humboldt-Universität zu Berlin, Berlin, Germany.

ABSTRACT
Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

Show MeSH
Related in: MedlinePlus