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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

Model of the TM4-Cx45CT construct. (A) Schematic diagram of full length Cx45. The black coloring represents the TM4-Cx45CT construct. (B) The amino acid sequence of the TM4-Cx45CT (D219-I396) contains a portion of the EL2 (line) and the entire TM4 (bold) and CT (arrow) domains.
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Figure 1: Model of the TM4-Cx45CT construct. (A) Schematic diagram of full length Cx45. The black coloring represents the TM4-Cx45CT construct. (B) The amino acid sequence of the TM4-Cx45CT (D219-I396) contains a portion of the EL2 (line) and the entire TM4 (bold) and CT (arrow) domains.

Mentions: TM4-CxCT domains used in this study were from the Cx50, Cx45, Cx43, Cx40, Cx37, Cx32, and Cx26 isoforms. Table 1 provides the amino acid sequence and species used for each TM4-CT domain to clone and ligate into the pET-14b expression vector (N-terminal 6× His-tag, ampicillin resistance; Novagen). Each construct includes 10 residues prior to their predicted TM4 domain (e.g., TM4-Cx45CT, Figure 1). All plasmid sequences were verified at the University of Nebraska Medical Center DNA Sequencing Core Facility.


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)

Model of the TM4-Cx45CT construct. (A) Schematic diagram of full length Cx45. The black coloring represents the TM4-Cx45CT construct. (B) The amino acid sequence of the TM4-Cx45CT (D219-I396) contains a portion of the EL2 (line) and the entire TM4 (bold) and CT (arrow) domains.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Model of the TM4-Cx45CT construct. (A) Schematic diagram of full length Cx45. The black coloring represents the TM4-Cx45CT construct. (B) The amino acid sequence of the TM4-Cx45CT (D219-I396) contains a portion of the EL2 (line) and the entire TM4 (bold) and CT (arrow) domains.
Mentions: TM4-CxCT domains used in this study were from the Cx50, Cx45, Cx43, Cx40, Cx37, Cx32, and Cx26 isoforms. Table 1 provides the amino acid sequence and species used for each TM4-CT domain to clone and ligate into the pET-14b expression vector (N-terminal 6× His-tag, ampicillin resistance; Novagen). Each construct includes 10 residues prior to their predicted TM4 domain (e.g., TM4-Cx45CT, Figure 1). All plasmid sequences were verified at the University of Nebraska Medical Center DNA Sequencing Core Facility.

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