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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

Western blot analysis. C41(DE3)pLysS transformed with the empty pET-14b vector (lanes 2 and 3) or the TM4-Cx45CT plasmid (lanes 5 and 6). Lane 8 contains the TM4-Cx45CT after purification. 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 (lanes 2 and 5) and 4 h after IPTG induction (lanes 3 and 6). 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. (A) Coomassie Blue stained gel is shown as a reference. Western blot analyses were performed using either (B) anti-His6, or (C) anti-Cx45 primary antibodies. The expected molecular mass of the TM4-Cx45CT is ~22 kDa, which is indicated by the arrow. Of note, in (B) lane 8, the anti-His6 primary antibody also reacted with a ~20 Da protein. Although not present in the (A) 15% SDS-PAGE gel or reactive with the (C) anti-Cx45 primary antibody, we speculate the doublet is caused by proteolysis of the TM4-Cx45CT.
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Figure 3: Western blot analysis. C41(DE3)pLysS transformed with the empty pET-14b vector (lanes 2 and 3) or the TM4-Cx45CT plasmid (lanes 5 and 6). Lane 8 contains the TM4-Cx45CT after purification. 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 (lanes 2 and 5) and 4 h after IPTG induction (lanes 3 and 6). 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. (A) Coomassie Blue stained gel is shown as a reference. Western blot analyses were performed using either (B) anti-His6, or (C) anti-Cx45 primary antibodies. The expected molecular mass of the TM4-Cx45CT is ~22 kDa, which is indicated by the arrow. Of note, in (B) lane 8, the anti-His6 primary antibody also reacted with a ~20 Da protein. Although not present in the (A) 15% SDS-PAGE gel or reactive with the (C) anti-Cx45 primary antibody, we speculate the doublet is caused by proteolysis of the TM4-Cx45CT.

Mentions: The pLysS (also referred to as pLysSRARE2) plasmid was isolated from Rosetta 2(DE3)pLysS cells using the QIAprep Spin Miniprep Kit (Qiagen) and co-transformed with the TM4-Cx45CT plasmid into the C41(DE3) and C43(DE3) strains. The cells were grown and induced as described above. The electrophoretic profile of total cellular proteins obtained 4 h after induction showed that TM4-Cx45CT expression in C43(DE3) was similar with and without transformation of the pLysS plasmid (Figure 2, lanes 7 and 11, respectively). Conversely, TM4-Cx45CT expression was significantly increased in the C41(DE3)pLysS strain as compared to C41(DE3) alone (Figure 2, lanes 9 and 5, respectively). The expression was quantified by densitometry and revealed that the addition of the pLysS plasmid increased TM4-Cx45CT expression by 70% in C41(DE3). The expression of TM4-Cx45CT was confirmed by Western blot analyses using anti-His6 and anti-Cx45 antibodies (Figure 3).


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)

Western blot analysis. C41(DE3)pLysS transformed with the empty pET-14b vector (lanes 2 and 3) or the TM4-Cx45CT plasmid (lanes 5 and 6). Lane 8 contains the TM4-Cx45CT after purification. 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 (lanes 2 and 5) and 4 h after IPTG induction (lanes 3 and 6). 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. (A) Coomassie Blue stained gel is shown as a reference. Western blot analyses were performed using either (B) anti-His6, or (C) anti-Cx45 primary antibodies. The expected molecular mass of the TM4-Cx45CT is ~22 kDa, which is indicated by the arrow. Of note, in (B) lane 8, the anti-His6 primary antibody also reacted with a ~20 Da protein. Although not present in the (A) 15% SDS-PAGE gel or reactive with the (C) anti-Cx45 primary antibody, we speculate the doublet is caused by proteolysis of the TM4-Cx45CT.
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Related In: Results  -  Collection

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Figure 3: Western blot analysis. C41(DE3)pLysS transformed with the empty pET-14b vector (lanes 2 and 3) or the TM4-Cx45CT plasmid (lanes 5 and 6). Lane 8 contains the TM4-Cx45CT after purification. 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 (lanes 2 and 5) and 4 h after IPTG induction (lanes 3 and 6). 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. (A) Coomassie Blue stained gel is shown as a reference. Western blot analyses were performed using either (B) anti-His6, or (C) anti-Cx45 primary antibodies. The expected molecular mass of the TM4-Cx45CT is ~22 kDa, which is indicated by the arrow. Of note, in (B) lane 8, the anti-His6 primary antibody also reacted with a ~20 Da protein. Although not present in the (A) 15% SDS-PAGE gel or reactive with the (C) anti-Cx45 primary antibody, we speculate the doublet is caused by proteolysis of the TM4-Cx45CT.
Mentions: The pLysS (also referred to as pLysSRARE2) plasmid was isolated from Rosetta 2(DE3)pLysS cells using the QIAprep Spin Miniprep Kit (Qiagen) and co-transformed with the TM4-Cx45CT plasmid into the C41(DE3) and C43(DE3) strains. The cells were grown and induced as described above. The electrophoretic profile of total cellular proteins obtained 4 h after induction showed that TM4-Cx45CT expression in C43(DE3) was similar with and without transformation of the pLysS plasmid (Figure 2, lanes 7 and 11, respectively). Conversely, TM4-Cx45CT expression was significantly increased in the C41(DE3)pLysS strain as compared to C41(DE3) alone (Figure 2, lanes 9 and 5, respectively). The expression was quantified by densitometry and revealed that the addition of the pLysS plasmid increased TM4-Cx45CT expression by 70% in C41(DE3). The expression of TM4-Cx45CT was confirmed by Western blot analyses using anti-His6 and anti-Cx45 antibodies (Figure 3).

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