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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

Secondary structure of the TM4-Cx45CT. Circular dichroïsm of the TM4-Cx45CT in 20 mM MES, 1 mM DTT, 1 mM EDTA, and 8% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] detergent micelles alone (black) or in presence of 30% acetonitrile (gray) at pH 7.5 (solid lines) and 5.8 (dashed lines).
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Figure 7: Secondary structure of the TM4-Cx45CT. Circular dichroïsm of the TM4-Cx45CT in 20 mM MES, 1 mM DTT, 1 mM EDTA, and 8% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] detergent micelles alone (black) or in presence of 30% acetonitrile (gray) at pH 7.5 (solid lines) and 5.8 (dashed lines).

Mentions: Circular dichro├»sm was used to gain insight into the TM4-Cx45CT secondary structure before obtaining an atomic level structure. Intracellular acidification is a major consequence of tissue ischemia during a myocardial infarction, which leads to closure and degradation of gap junction channels and can be a substrate for malignant ventricular arrhythmias (Lau, 2005). Therefore, data were collected at either physiological (pH 7.5) or ischemic (pH 5.8) conditions (Figure 7). The TM4-Cx45CT (without acetonitrile) has a small increase in ╬▒-helical content under acidic conditions (pH 7.5, 25%; pH 5.8, 28%). This pH-effect is similar in the presence of 30% acetonitrile with a small increase in overall ╬▒-helical content (pH 7.5 28%; pH 5.8, 32%). The pH-induced increase in ╬▒-helical content for the TM4-Cx45CT (3ÔÇô4%) is smaller than observed for the TM4-Cx43CT (16%; Kellezi et al., 2008; Grosely et al., 2012).


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)

Secondary structure of the TM4-Cx45CT. Circular dichroïsm of the TM4-Cx45CT in 20 mM MES, 1 mM DTT, 1 mM EDTA, and 8% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] detergent micelles alone (black) or in presence of 30% acetonitrile (gray) at pH 7.5 (solid lines) and 5.8 (dashed lines).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Secondary structure of the TM4-Cx45CT. Circular dichroïsm of the TM4-Cx45CT in 20 mM MES, 1 mM DTT, 1 mM EDTA, and 8% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] detergent micelles alone (black) or in presence of 30% acetonitrile (gray) at pH 7.5 (solid lines) and 5.8 (dashed lines).
Mentions: Circular dichro├»sm was used to gain insight into the TM4-Cx45CT secondary structure before obtaining an atomic level structure. Intracellular acidification is a major consequence of tissue ischemia during a myocardial infarction, which leads to closure and degradation of gap junction channels and can be a substrate for malignant ventricular arrhythmias (Lau, 2005). Therefore, data were collected at either physiological (pH 7.5) or ischemic (pH 5.8) conditions (Figure 7). The TM4-Cx45CT (without acetonitrile) has a small increase in ╬▒-helical content under acidic conditions (pH 7.5, 25%; pH 5.8, 28%). This pH-effect is similar in the presence of 30% acetonitrile with a small increase in overall ╬▒-helical content (pH 7.5 28%; pH 5.8, 32%). The pH-induced increase in ╬▒-helical content for the TM4-Cx45CT (3ÔÇô4%) is smaller than observed for the TM4-Cx43CT (16%; Kellezi et al., 2008; Grosely et al., 2012).

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