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An Escherichia coli strain for expression of the connexin45 carboxyl terminus attached to the 4th transmembrane domain.

Kopanic JL, Al-Mugotir M, Zach S, Das S, Grosely R, Sorgen PL - Front Pharmacol (2013)

Bottom Line: This occurs because proteins must be expressed in minimal based media.Here, we describe an expression system for membrane proteins that significantly improves yield by addressing two common problems, cell toxicity caused by protein translation and codon bias between genomes.This work provides researchers with a cost-effective tool for NMR and other biophysical studies, to use when faced with little-to-no expression of eukaryotic membrane proteins in Escherichia coli expression systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center Omaha, NE, USA.

ABSTRACT
A major problem for structural characterization of membrane proteins, such as connexins, by nuclear magnetic resonance (NMR) occurs at the initial step of the process, the production of sufficient amounts of protein. This occurs because proteins must be expressed in minimal based media. Here, we describe an expression system for membrane proteins that significantly improves yield by addressing two common problems, cell toxicity caused by protein translation and codon bias between genomes. This work provides researchers with a cost-effective tool for NMR and other biophysical studies, to use when faced with little-to-no expression of eukaryotic membrane proteins in Escherichia coli expression systems.

No MeSH data available.


Related in: MedlinePlus

Comparison of the TM4-Cx45CT expression profile obtained from E. coli strain C41(DE3) with the pLysS plasmid in LB and minimal media. C41(DE3)pLysS transformed with the TM4-Cx45CT plasmid was grown in LB medium (lanes 2 and 3), 15N-labeled M63 minimal medium (lanes 5 and 6), and 15N-labeled M63 minimal medium supplemented with 1 g 15N-ISOGRO/L medium (lanes 8 and 9). Lane 1 contains the Precision Plus Protein All Blue Standards molecular mass marker (Bio-Rad) and lanes 4 and 7 are blank. Samples were collected just prior to (uninduced, U) and 4 h after IPTG induction (I), as noted. A total of 500 μL samples with an Abs600nm at 0.5 were pelleted and resuspended in 30 μL of 6× SDS loading buffer. Equal amounts of total protein (7 μL) were ran on a 15% SDS-PAGE gel and stained with Coomassie Blue. The TM4-Cx45CT has an expected molecular mass of ~22 kDa, indicated by the arrow.
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Figure 4: Comparison of the TM4-Cx45CT expression profile obtained from E. coli strain C41(DE3) with the pLysS plasmid in LB and minimal media. C41(DE3)pLysS transformed with the TM4-Cx45CT plasmid was grown in LB medium (lanes 2 and 3), 15N-labeled M63 minimal medium (lanes 5 and 6), and 15N-labeled M63 minimal medium supplemented with 1 g 15N-ISOGRO/L medium (lanes 8 and 9). Lane 1 contains the Precision Plus Protein All Blue Standards molecular mass marker (Bio-Rad) and lanes 4 and 7 are blank. Samples were collected just prior to (uninduced, U) and 4 h after IPTG induction (I), as noted. A total of 500 μL samples with an Abs600nm at 0.5 were pelleted and resuspended in 30 μL of 6× SDS loading buffer. Equal amounts of total protein (7 μL) were ran on a 15% SDS-PAGE gel and stained with Coomassie Blue. The TM4-Cx45CT has an expected molecular mass of ~22 kDa, indicated by the arrow.

Mentions: Expression using the C41(DE3)pLysS strain was tested in isotopically labeled M63 minimal medium, which allows the control of nitrogen and carbon sources needed for NMR structural studies. The TM4-Cx45CT expression level decreased 84% in M63 as compared to LB (Figure 4, lanes 6 and 3, respectively). However, expression was restored to LB level when M63 was supplemented with 15N-ISOGRO (1 g/L, Isotec; Figure 4, lane 9). ISOGRO is an algal lysate-derived complex labeling medium that provides cells a metabolic boost that often decreases lag time, facilitates the attainment of growth saturation, and promotes recombinant protein production. ISOGRO helps cultures conserve cellular energy by limiting the requirement for de novo synthesis of cellular machinery and metabolic precursors; permitting more cellular resources to be direct toward recombinant protein expression. At this expression level, 8 L of growth is necessary to obtain a 1 mM at 300 μL volume (“gold standard” concentration and volume for NMR structural studies; Table 1). This is in contrast to the 54 or 180 L would be necessary in M63 without 15N-ISOGRO or without 15N-ISOGRO and the pLysS plasmid, respectively.


An Escherichia coli strain for expression of the connexin45 carboxyl terminus attached to the 4th transmembrane domain.

Kopanic JL, Al-Mugotir M, Zach S, Das S, Grosely R, Sorgen PL - Front Pharmacol (2013)

Comparison of the TM4-Cx45CT expression profile obtained from E. coli strain C41(DE3) with the pLysS plasmid in LB and minimal media. C41(DE3)pLysS transformed with the TM4-Cx45CT plasmid was grown in LB medium (lanes 2 and 3), 15N-labeled M63 minimal medium (lanes 5 and 6), and 15N-labeled M63 minimal medium supplemented with 1 g 15N-ISOGRO/L medium (lanes 8 and 9). Lane 1 contains the Precision Plus Protein All Blue Standards molecular mass marker (Bio-Rad) and lanes 4 and 7 are blank. Samples were collected just prior to (uninduced, U) and 4 h after IPTG induction (I), as noted. A total of 500 μL samples with an Abs600nm at 0.5 were pelleted and resuspended in 30 μL of 6× SDS loading buffer. Equal amounts of total protein (7 μL) were ran on a 15% SDS-PAGE gel and stained with Coomassie Blue. The TM4-Cx45CT has an expected molecular mass of ~22 kDa, indicated by the arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750199&req=5

Figure 4: Comparison of the TM4-Cx45CT expression profile obtained from E. coli strain C41(DE3) with the pLysS plasmid in LB and minimal media. C41(DE3)pLysS transformed with the TM4-Cx45CT plasmid was grown in LB medium (lanes 2 and 3), 15N-labeled M63 minimal medium (lanes 5 and 6), and 15N-labeled M63 minimal medium supplemented with 1 g 15N-ISOGRO/L medium (lanes 8 and 9). Lane 1 contains the Precision Plus Protein All Blue Standards molecular mass marker (Bio-Rad) and lanes 4 and 7 are blank. Samples were collected just prior to (uninduced, U) and 4 h after IPTG induction (I), as noted. A total of 500 μL samples with an Abs600nm at 0.5 were pelleted and resuspended in 30 μL of 6× SDS loading buffer. Equal amounts of total protein (7 μL) were ran on a 15% SDS-PAGE gel and stained with Coomassie Blue. The TM4-Cx45CT has an expected molecular mass of ~22 kDa, indicated by the arrow.
Mentions: Expression using the C41(DE3)pLysS strain was tested in isotopically labeled M63 minimal medium, which allows the control of nitrogen and carbon sources needed for NMR structural studies. The TM4-Cx45CT expression level decreased 84% in M63 as compared to LB (Figure 4, lanes 6 and 3, respectively). However, expression was restored to LB level when M63 was supplemented with 15N-ISOGRO (1 g/L, Isotec; Figure 4, lane 9). ISOGRO is an algal lysate-derived complex labeling medium that provides cells a metabolic boost that often decreases lag time, facilitates the attainment of growth saturation, and promotes recombinant protein production. ISOGRO helps cultures conserve cellular energy by limiting the requirement for de novo synthesis of cellular machinery and metabolic precursors; permitting more cellular resources to be direct toward recombinant protein expression. At this expression level, 8 L of growth is necessary to obtain a 1 mM at 300 μL volume (“gold standard” concentration and volume for NMR structural studies; Table 1). This is in contrast to the 54 or 180 L would be necessary in M63 without 15N-ISOGRO or without 15N-ISOGRO and the pLysS plasmid, respectively.

Bottom Line: This occurs because proteins must be expressed in minimal based media.Here, we describe an expression system for membrane proteins that significantly improves yield by addressing two common problems, cell toxicity caused by protein translation and codon bias between genomes.This work provides researchers with a cost-effective tool for NMR and other biophysical studies, to use when faced with little-to-no expression of eukaryotic membrane proteins in Escherichia coli expression systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center Omaha, NE, USA.

ABSTRACT
A major problem for structural characterization of membrane proteins, such as connexins, by nuclear magnetic resonance (NMR) occurs at the initial step of the process, the production of sufficient amounts of protein. This occurs because proteins must be expressed in minimal based media. Here, we describe an expression system for membrane proteins that significantly improves yield by addressing two common problems, cell toxicity caused by protein translation and codon bias between genomes. This work provides researchers with a cost-effective tool for NMR and other biophysical studies, to use when faced with little-to-no expression of eukaryotic membrane proteins in Escherichia coli expression systems.

No MeSH data available.


Related in: MedlinePlus