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High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel.

Maduke M, Pheasant DJ, Miller C - J. Gen. Physiol. (1999)

Bottom Line: ClC-type anion-selective channels are widespread throughout eukaryotic organisms.BLAST homology searches reveal that many microbial genomes also contain members of the ClC family.Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02254-9110, USA.

ABSTRACT
ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli-derived ClC Cl(-) channel homologue, "EriC," the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.

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Overexpression and purification of EriC. Protein samples were run on a 13% polyacrylamide gel. (1) Molecular mass markers (broad range; Bio-Rad Laboratories), (2) control membranes (600 μg protein, estimated as 0.6 A280-ml) from E. coli induced to express KcsA, (3) membranes from E. coli expressing EriC (600 μg protein), (4) clarified membrane extract (equivalent to 600-μg membranes), (5) 9 μg purified EriC.
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Figure 2: Overexpression and purification of EriC. Protein samples were run on a 13% polyacrylamide gel. (1) Molecular mass markers (broad range; Bio-Rad Laboratories), (2) control membranes (600 μg protein, estimated as 0.6 A280-ml) from E. coli induced to express KcsA, (3) membranes from E. coli expressing EriC (600 μg protein), (4) clarified membrane extract (equivalent to 600-μg membranes), (5) 9 μg purified EriC.

Mentions: We cloned one of the above ClC genes into an E. coli expression vector and added an NH2-terminal hexahistidine sequence for large-scale production and straightforward purification of EriC, a putative ClC-type channel product of the yadQ gene. After an overnight room-temperature induction of E. coli bearing the pEriC plasmid, a protein band of ∼38 kD, absent in uninduced controls, was observed on SDS-PAGE of bacterial membrane fractions (Fig. 2). This protein was detergent-extracted from the bacterial membrane fraction and purified on a Ni-chelate column. The apparent molecular mass of ∼38 kD is smaller than that calculated from the sequence (51 kD), but the integrity of the full-length protein is indicated by two observations. First, robust interaction with the metal-affinity column indicates that the NH2-terminal hexahistidine tag is present; second, an EriC construct carrying an eight-residue, COOH-terminal 1D4 epitope (Molday and MacKenzie 1983), showed a similar apparent molecular mass, detected on immunoblots (data not shown). Typical yields of purified EriC are 0.5–1 mg/liter culture.


High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel.

Maduke M, Pheasant DJ, Miller C - J. Gen. Physiol. (1999)

Overexpression and purification of EriC. Protein samples were run on a 13% polyacrylamide gel. (1) Molecular mass markers (broad range; Bio-Rad Laboratories), (2) control membranes (600 μg protein, estimated as 0.6 A280-ml) from E. coli induced to express KcsA, (3) membranes from E. coli expressing EriC (600 μg protein), (4) clarified membrane extract (equivalent to 600-μg membranes), (5) 9 μg purified EriC.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230540&req=5

Figure 2: Overexpression and purification of EriC. Protein samples were run on a 13% polyacrylamide gel. (1) Molecular mass markers (broad range; Bio-Rad Laboratories), (2) control membranes (600 μg protein, estimated as 0.6 A280-ml) from E. coli induced to express KcsA, (3) membranes from E. coli expressing EriC (600 μg protein), (4) clarified membrane extract (equivalent to 600-μg membranes), (5) 9 μg purified EriC.
Mentions: We cloned one of the above ClC genes into an E. coli expression vector and added an NH2-terminal hexahistidine sequence for large-scale production and straightforward purification of EriC, a putative ClC-type channel product of the yadQ gene. After an overnight room-temperature induction of E. coli bearing the pEriC plasmid, a protein band of ∼38 kD, absent in uninduced controls, was observed on SDS-PAGE of bacterial membrane fractions (Fig. 2). This protein was detergent-extracted from the bacterial membrane fraction and purified on a Ni-chelate column. The apparent molecular mass of ∼38 kD is smaller than that calculated from the sequence (51 kD), but the integrity of the full-length protein is indicated by two observations. First, robust interaction with the metal-affinity column indicates that the NH2-terminal hexahistidine tag is present; second, an EriC construct carrying an eight-residue, COOH-terminal 1D4 epitope (Molday and MacKenzie 1983), showed a similar apparent molecular mass, detected on immunoblots (data not shown). Typical yields of purified EriC are 0.5–1 mg/liter culture.

Bottom Line: ClC-type anion-selective channels are widespread throughout eukaryotic organisms.BLAST homology searches reveal that many microbial genomes also contain members of the ClC family.Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02254-9110, USA.

ABSTRACT
ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli-derived ClC Cl(-) channel homologue, "EriC," the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.

Show MeSH
Related in: MedlinePlus