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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

TM4-Cx45CT expression profile obtained from different E. coli (DE3) strains with and without the pLysS plasmid in LB medium. TM4-Cx45CT was expressed in the following E. coli cell strains: BL21(DE3) (lanes 2 and 3), C41(DE3) (lanes 4 and 5), C43(DE3) (lanes 6 and 7), C41(DE3)pLysS (lanes 8 and 9), C43(DE3)pLysS (lanes 10 and 11), and Rosetta 2(DE3)pLysS (lanes 12 and 13). Lanes 1 and 14 contain the Precision Plus Protein All Blue Standards molecular mass marker (Bio-Rad). Samples were collected just prior to (uninduced; U) and 4 h after IPTG induction (I), as noted. All lanes contain an equal amount of total protein for comparison. This was accomplished by pelleting 500 μL samples with an Abs600nm at 0.5 and resuspending the pellet in 30 μL of 6× SDS loading buffer. Five microliters of the samples 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 2: TM4-Cx45CT expression profile obtained from different E. coli (DE3) strains with and without the pLysS plasmid in LB medium. TM4-Cx45CT was expressed in the following E. coli cell strains: BL21(DE3) (lanes 2 and 3), C41(DE3) (lanes 4 and 5), C43(DE3) (lanes 6 and 7), C41(DE3)pLysS (lanes 8 and 9), C43(DE3)pLysS (lanes 10 and 11), and Rosetta 2(DE3)pLysS (lanes 12 and 13). Lanes 1 and 14 contain the Precision Plus Protein All Blue Standards molecular mass marker (Bio-Rad). Samples were collected just prior to (uninduced; U) and 4 h after IPTG induction (I), as noted. All lanes contain an equal amount of total protein for comparison. This was accomplished by pelleting 500 μL samples with an Abs600nm at 0.5 and resuspending the pellet in 30 μL of 6× SDS loading buffer. Five microliters of the samples 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: The E. coli strains BL21(DE3), C41(DE3), C43(DE3), and Rosetta 2(DE3)pLysS (chloramphenicol resistance) were transformed with the TM4-Cx45CT plasmid and incubated in lysogeny broth (LB) medium at 37°C, 250 rpm. Rosetta 2(DE3)pLysS expresses seven rare codons (Arg, AGA, AGG, CGA, CGG; Ile, AUA; Pro, CCC, Leu, CUA) in comparison to the BL21-Codon Plus(DE3)-RIPL strain (contains the most amount of tRNA genes in the BL21-Codon Plus series), which contains only five tRNA genes (Arg, AGA, AGG; Ile, AUA; Pro, CCC, Leu, CUA). The TM4-Cx45CT contains five Arg rare codons, including two CGA, which the BL21-Codon Plus(DE3)-RIPL strain does not have the corresponding tRNA. Therefore, Rosetta 2(DE3)pLysS was chosen as the representative strain that expresses rare codons for this study. Protein expression was induced by the addition of 1.0 mM IPTG (final concentration; Bioexpress) at an optical density of 0.6 at 600 nm. The electrophoretic profile of total cellular proteins obtained 4 h after induction indicated that only C41(DE3) and C43(DE3) expressed TM4-Cx45CT (Figure 2, lanes 5 and 7, respectively). However, this protein yield is not optimal as significantly less protein per liter was produced then what was needed for the TM4-Cx43CT NMR structural studies (Kellezi et al., 2008). Upon examination of the TM4-Cx45CT gene sequence, 14 rare codons were identified; of these, eight (two in tandem) translate to the amino acid with the greatest codon bias, Arg (Table 2). Even though the Rosetta 2(DE3)pLysS strain was unable to express TM4-Cx45CT (Figure 2, lane 13), we hypothesized that expression would be enhanced by combining the pLysS plasmid with the C41(DE3) or C43(DE3) strains.


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)

TM4-Cx45CT expression profile obtained from different E. coli (DE3) strains with and without the pLysS plasmid in LB medium. TM4-Cx45CT was expressed in the following E. coli cell strains: BL21(DE3) (lanes 2 and 3), C41(DE3) (lanes 4 and 5), C43(DE3) (lanes 6 and 7), C41(DE3)pLysS (lanes 8 and 9), C43(DE3)pLysS (lanes 10 and 11), and Rosetta 2(DE3)pLysS (lanes 12 and 13). Lanes 1 and 14 contain the Precision Plus Protein All Blue Standards molecular mass marker (Bio-Rad). Samples were collected just prior to (uninduced; U) and 4 h after IPTG induction (I), as noted. All lanes contain an equal amount of total protein for comparison. This was accomplished by pelleting 500 μL samples with an Abs600nm at 0.5 and resuspending the pellet in 30 μL of 6× SDS loading buffer. Five microliters of the samples 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

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Figure 2: TM4-Cx45CT expression profile obtained from different E. coli (DE3) strains with and without the pLysS plasmid in LB medium. TM4-Cx45CT was expressed in the following E. coli cell strains: BL21(DE3) (lanes 2 and 3), C41(DE3) (lanes 4 and 5), C43(DE3) (lanes 6 and 7), C41(DE3)pLysS (lanes 8 and 9), C43(DE3)pLysS (lanes 10 and 11), and Rosetta 2(DE3)pLysS (lanes 12 and 13). Lanes 1 and 14 contain the Precision Plus Protein All Blue Standards molecular mass marker (Bio-Rad). Samples were collected just prior to (uninduced; U) and 4 h after IPTG induction (I), as noted. All lanes contain an equal amount of total protein for comparison. This was accomplished by pelleting 500 μL samples with an Abs600nm at 0.5 and resuspending the pellet in 30 μL of 6× SDS loading buffer. Five microliters of the samples 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: The E. coli strains BL21(DE3), C41(DE3), C43(DE3), and Rosetta 2(DE3)pLysS (chloramphenicol resistance) were transformed with the TM4-Cx45CT plasmid and incubated in lysogeny broth (LB) medium at 37°C, 250 rpm. Rosetta 2(DE3)pLysS expresses seven rare codons (Arg, AGA, AGG, CGA, CGG; Ile, AUA; Pro, CCC, Leu, CUA) in comparison to the BL21-Codon Plus(DE3)-RIPL strain (contains the most amount of tRNA genes in the BL21-Codon Plus series), which contains only five tRNA genes (Arg, AGA, AGG; Ile, AUA; Pro, CCC, Leu, CUA). The TM4-Cx45CT contains five Arg rare codons, including two CGA, which the BL21-Codon Plus(DE3)-RIPL strain does not have the corresponding tRNA. Therefore, Rosetta 2(DE3)pLysS was chosen as the representative strain that expresses rare codons for this study. Protein expression was induced by the addition of 1.0 mM IPTG (final concentration; Bioexpress) at an optical density of 0.6 at 600 nm. The electrophoretic profile of total cellular proteins obtained 4 h after induction indicated that only C41(DE3) and C43(DE3) expressed TM4-Cx45CT (Figure 2, lanes 5 and 7, respectively). However, this protein yield is not optimal as significantly less protein per liter was produced then what was needed for the TM4-Cx43CT NMR structural studies (Kellezi et al., 2008). Upon examination of the TM4-Cx45CT gene sequence, 14 rare codons were identified; of these, eight (two in tandem) translate to the amino acid with the greatest codon bias, Arg (Table 2). Even though the Rosetta 2(DE3)pLysS strain was unable to express TM4-Cx45CT (Figure 2, lane 13), we hypothesized that expression would be enhanced by combining the pLysS plasmid with the C41(DE3) or C43(DE3) strains.

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